Transgenic plants expressing a pH-sensitive nitrate transporter

ABSTRACT

The invention relates to transgenic plants with improved growth and nitrogen use efficiency expressing nitrate transporter gene, methods of making such plants and methods for improving growth and nitrogen use efficiency.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. application Ser. No.14/642,858, filed Mar. 3, 2015, which is a continuation of U.S.application Ser. No. 14/172,294, filed Feb. 4, 2014, now U.S. Pat. No.9,012,721, Issued Apr. 21, 2015, which is a continuation-in-partapplication of international patent application Serial No.PCT/CN2013/071384 filed Feb. 5, 2013.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The invention relates to transgenic plants with improved traits, forexample growth and nitrogen use efficiency expressing a nitratetransporter gene, methods of making such plants and methods forimproving growth and nitrogen use efficiency.

INTRODUCTION

Global crop productivity has increased markedly during the past fivedecades mainly due to improved crop varieties and massive inputs ofchemical fertilizers, especially nitrogen (N)^(1,2). However, fertilizerN use efficiency is only about 30-50% for many crops²⁻⁴ with largeproportions being lost to the environment, resulting in variousdetrimental impacts such as the degradation of air and water quality andlosses of biodiversity^(5,6). It has been estimated that excess N in theenvironment is currently costing the European Union between

70 billion and

320 billion per year⁷. In China, the increase in grain production duringthe past 30 years has been accompanied by a dramatic decrease in the Nuse efficiency (NUE) from 55 to 20 kg grain per kg fertilizer Napplied³. In Asia, rice provides more than 70% of the daily caloricintake of the population, but with the land available for agriculturediminishing, increasing demand can only be managed by increasingproductivity.

It is therefore of major importance to identify the critical stepscontrolling plant NUE. NUE can be defined as being the yield of grainper unit of available N in the soil (including the residual N present inthe soil and the fertilizer). Thus NUE can be divided into twoprocesses: uptake efficiency (NupE; the ability of the plant to remove Nfrom the soil as nitrate and ammonium ions) and the utilizationefficiency (NutE; the ability to use N to produce grain yield). Thischallenge is particularly relevant to cereals for which large amounts ofN fertilizers are required to attain maximum yield and for which NUE isestimated to be far less than 50% (Hirel et al).

Nitrogen (N) is fundamental to crop development as it forms the basiccomponent of many organic molecules, nucleic acids and proteins. Nnutrition affects all levels of plant function, from metabolism toresource allocation, growth, and development. The most abundant sourcefor N acquisition by plant roots is nitrate (NO₃ ⁻) in natural aerobicsoils, due to intensive nitrification of applied organic and fertilizerN. By contrast, ammonium (NH₄ ⁺) is the main form of available N inflooded paddy soils due to the anaerobic soil conditions (Sasakawa andYamamoto, 1978).

Thus, soil inorganic nitrogen (N) is predominantly available for plantsas nitrate in aerobic uplands and well-drained soils and as ammonium inpoorly drained soils and flooded anaerobic paddy fields. In many plantsthe nitrate acquired by roots is transported to the shoots before beingassimilated (Smirnoff and Stewart, 1985). By contrast, ammonium derivedfrom nitrate reduction or directly from ammonium uptake ispreferentially assimilated in the root and then transported in anorganic form to the shoot (Xu et al., 2012). To cope with variedconcentrations of nitrate in soils, plant roots have developed at leastthree nitrate uptake systems, two high-affinity transport systems (HATS)and one low-64 affinity transport system (LATS), responsible for theacquisition of nitrate (Crawford and Glass, 1998). The constitutive HATS(cHATS) and nitrate-inducible HATS (iHATS) operate to take up nitrate atlow nitrate concentration in external medium with saturation in a rangeof 0.2-0.5 mM. In contrast, LATS functions in nitrate acquisition athigher external nitrate 68 concentration. The uptake by LATS and HATS ismediated by nitrate transporters belonging to the families of NRT1 andNRT2, respectively (Forde, 2000; Miller et al., 2007). Uptake by rootsis regulated by negative feedback, linking the expression and activityof nitrate uptake to the N status of the plant (Miller et al., 2007).Several different N metabolites have been proposed to be cellularsensors of N status, including glutamine (Fan et al., 2006; Miller etal., 2008) and one model has root vacuolar nitrate as the feedbacksignal as these pools increase with plant N status.

Although higher plants have the capacity to utilize organic N, the majorsources for N acquisition by roots are considered to be NO₃ ⁻ and NH₄.Plants vary substantially in their relative adaptations to these twosources of N. Although NH₄ should be the preferred N source, since itsmetabolism requires less energy than that of NO₃ ⁻, only a few speciesactually perform well when NH₄ is provided as the only N source. Amongthe latter are boreal conifers, ericaceous species, some vegetablecrops, and rice (Oryza sativa L.). In contrast to these species, mostagricultural species develop at times severe toxicity symptoms on NH₄thus, superior growth in these species is seen on NO₃ ⁻. However, whenboth N sources are provided simultaneously, growth and yield are oftenenhanced significantly compared with growth on either NH₄ or NO₃ ⁻ alone(Kronzucker et al., 1999).

Rice, a major crop feeding almost 50% of the world's populationtherefore differs from other crop plants in that it is capable ofgrowing exclusively on NH₄ as the only N source. Rice has beentraditionally cultivated under flooded anaerobic soil conditions whereammonium is the main N source. However, the specialized aerenchyma cellsin rice roots can transfer oxygen from the shoots to the roots andrelease it to the rhizosphere, where bacterial conversion of ammonium tonitrate (nitrification) can take place⁸. Nitrification in thewaterlogged paddy rhizosphere can result in 25-40% of the total crop Nbeing taken up in the form of nitrate, mainly through a high affinitytransport system (HATS)⁹. The uptake of nitrate is mediated bycotransport with protons (H⁺) that can be extruded from the cell byplasma membrane H⁺-ATPases¹⁰. The molecular mechanisms of nitrate uptakeand translocation in rice are not fully understood. Since the nitrateconcentration in the rhizosphere of paddy fields is estimated to be lessthan 10 μM (Kirk and Kronzucker, 2005), NRT2 family members play a majorrole in nitrate uptake in rice (Araki and Hasegawa, 2006; Yan et al.,2011). In addition, rice roots have abundant aerenchyma for thetransportation of oxygen into the rhizosphere, resulting in ammoniumnitrification by bacteria on the root surface (Kirk, 2003; Li et al.,2008). Therefore, up to 40% of the total N taken up by rice roots grownunder wetland conditions might be in the form of nitrate and the ratesof uptake could be comparable with those of ammonium (Kronzucker et al.,2000; Kirk and Kronzucker, 2005).

Both electrophysiological and molecular studies have shown that nitrateuptake through both HATS and LATS is an active process mediated byproton/nitrate co-transporters (Zhou et al., 2000; Miller et al., 2007).In the Arabidopsis genome, there are at least 53 and 7 members belongingto NRT1 and NRT2 families, respectively (Miller et al., 2007; Tsay etal., 2007). Several Arabidopsis NRT1 and NRT2 family members have beencharacterized for their functions in nitrate uptake and long distancetransport. AtNRT1.1 (CHL1) is described as a transceptor playingmultiple roles as a dual affinity nitrate transporter and a sensor ofexternal nitrate supply concentration (Liu and Tsay, 2003; Ho et al.,2009; Gojon et al., 2011), and auxin transport at low nitrateconcentrations (Krouk et al., 2010). In contrast, AtNRT1.2 (NTL1) is aconstitutively expressed low affinity nitrate transporter (Huang et al.,1999). AtNRT1.4 is a leaf petiole expressed nitrate transporter andplays a critical role in regulating leaf nitrate homeostasis and leafdevelopment (Chiu et al., 2004). AtNRT1.5 is expressed in the rootpericycle cells close to the xylem and is responsible for loading ofnitrate into the xylem for root-to-shoot nitrate transport (Lin et al.,2008). AtNRT1.6 is expressed only in reproductive tissues and isinvolved in delivering nitrate from maternal tissue to the earlydeveloping embryo (Almagro et al., 2008). AtNRT1.7 functions in phloemloading of nitrate to allow transport out of older leaves and intoyounger leaves, indicating that source-to-sink remobilization of nitrateis mediated by the phloem (Fan et al., 2009). AtNRT1.8 is expressedpredominantly in xylem parenchyma cells within the vasculature and playsthe role in retrieval of nitrate from the xylem sap (Li et al., 2010).AtNRT1.9 facilitates loading of nitrate into the root phloem, enhancingdownward transport in roots, and its knockout increases root to shootxylem transport of nitrate (Wang and Tsay, 2011).

Among the 7 NRT2 family members in Arabidopsis, both AtNRT2.1 andAtNRT2.2 have been characterized as contributors to iHATS (Filleur etal., 2001). In addition, NRT2.1 transport activity requires a secondaccessory protein NAR2.1 (or NRT3.1) in Arabidopsis (Okamoto et al.,2006; Orsel et al., 2006; Yong et al., 2010). Knockout of AtNAR2.1(atnar2.1 mutant) had more severe effects on both nitrate uptake at lownitrate concentrations and growth than knockout of its partner AtNRT2.1(atnrt2.1 mutant) suggesting other functions for AtNAR2.1 (Orsel et al.,2006). Interestingly, AtNRT2.7 is expressed specifically in the vacuolarmembrane of reproductive organs and controls nitrate content in seeds(Chopin et al., 2007). Recently, AtNRT2.4 has been found to be a highaffinity plasma membrane nitrate transporter expressed in the epidermisof lateral roots and in or close to the shoot phloem (Kiba et al.,2012). AtNRT2.4 is involved in the uptake of NO₃ ⁻ by the root at verylow external concentration and in shoot NO₃ ⁻ loading into the phloemand is important under N starvation (Kiba et al., 2012).

In the rice genome, five NRT2 genes have been identified (Araki andHasegawa, 2006; Cai et al., 2008; Feng et al., 2011). OsNRT2.1 andOsNRT2.2 share an identical coding region sequence with different 5′-and 3′-untranscribed regions (UTRs) and have high similarity to the NRT2genes of other monocotyledons, while OsNRT2.3 and OsNRT2.4 are moreclosely related to Arabidopsis NRT2 genes. OsNRT2.3 mRNA is actuallyspliced into two gene products, OsNRT2.3a (AK109776) and OsNRT2.3b(AK072215), with 94.2% similarity in their putative amino acid sequences(Feng et al., 2011, Yan et al., 2011). OsNRT2.3a is expressed mainly inroots and this pattern is enhanced by nitrate supply, while OsNRT2.3b isexpressed weakly in roots and relatively abundantly in shoots with noeffect of the N form and concentration on the amount of transcript (Fenget al., 2011, Feng 2012).

CN101392257 shows an expression analysis of OsNRT2.3a and b in rice andXenopus oocytes and mentions overexpression of the OsNRT2.3 gene inplants. CN101392257 does not disclose separate expression of OsNRT2.3aand OsNRT2.3b in rice nor does it show that expressing OsNRT2.3b inplants other than rice which significantly differ in the use of Nsources can have beneficial effects.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There is a need to provide more nutrient efficient genotypes for cropplants to ensure sustainable crop production for global food securityand to reduce the costs and negative environmental effects of mineralfertiliser input, such as of air and water quality and losses ofbiodiversity. The present invention is aimed at addressing this need.

The rice transporter OsNRT2.3 has two spliced forms. Some nitratetransporters require two genes for function; the second much smallercomponent (OsNAR21) is required for the correct targeting of thetransporter protein to the plasma membrane. One of the two splicedforms, OsNRT2.3a, requires this second component for function, while theother form, OsNRT2.3b, does not. Applicants have demonstrated for thefirst time that expression of both nitrate transporters in Xenopusoocytes showed that only OsNRT2.3b had a pH-sensitive regulatory site onthe cytoplasmic face of the protein. This pH sensing site was confirmedby site-directed mutagenesis of a histidine amino acid residue (H167R)in the pH sensing motif. In rice, OsNRT2.3b was more specificallylocalised in the vascular tissue, particularly the phloem. Applicantstherefore suggest that the protein is specifically involved in longdistance transport within the plant and that the phloem is important inwhole plant pH regulation.

Applicants have over-expressed, independently, both OsNRT2.3a/b genesand the H167R mutated form of OsNRT2.3b using strong non-specificconstitutive promoters (35S and ubiquitin) in several different Chinesecultivars of rice. Applicants have shown that only OsNRT2.3bover-expressing plants showed much improved growth and nitrogen useefficiency and the phenotype was surprising as both nitrate and ammoniumuptake was increased in these OsNRT2.3b over-expressing plants. TheOsNRT2.3b over-expressing plants showed less photorespiration andgenerally had better pH regulation (iron and phosphate contents)relative to controls or OsNRT2.3a over-expressing plants. The pH sensingmotif of OsNRT2.3b is important for these effects in rice by linking theplant's pH status to nitrate supply.

Applicants have also surprisingly shown that OsNRT2.3b is functionalwhen transgenically expressed in plants other than rice although theseplants, such as Arabidopsis, wheat and tobacco, differ fundamentally intheir use of nitrogen sources.

As can be seen from the following disclosure, the invention has severalaspects. In some aspects, the invention relates to methods, uses andplants where rice is specifically disclaimed. In other aspects, theinvention relates to methods, uses and plants where the expression ofthe OsNRT2.3b nucleic acid is regulated by a phloem specific promoter.In other aspects, the invention relates to methods, uses and plants thatdo not transgenically express a nucleic acid sequence which may compriseSEQ ID No. 2 or a functional variant thereof.

Thus, in a first aspect, the invention relates to methods for increasingone or more of growth, yield, nitrogen transport, NUE, nitrogenacquisition, decreasing photorespiration, increasing intercellular CO₂levels, increasing photosynthetic efficiency, pathogen resistance,survival and maintaining/improving pH homeostasis which may compriseintroducing and expressing a nucleic acid construct which may compriseSEQ ID No. 1, a functional variant, part or homolog thereof operablylinked to a regulatory sequence in a plant.

In a second aspect, the invention relates to a method for increasing oneor more of growth, yield, nitrogen use efficiency, nitrogen transport,nitrogen stress tolerance, pathogen resistance, survival and/or nitrogenacquisition of a plant which may comprise introducing and expressing anucleic acid construct which may comprise a nucleic acid sequence asdefined in SEQ ID No. 1, a functional variant, part or homolog thereofoperably linked to a regulatory sequence in a plant wherein if thenucleic acid sequence is as defined in SEQ ID No. 1, a functionalvariant, part or homolog thereof said plant is not rice.

In a third aspect, the invention relates to a transgenic plantexpressing a nucleic acid construct which may comprise a nucleic acidsequence as defined in SEQ ID No. 1, a functional variant, part orhomolog thereof operably linked to a regulatory sequence into a plantwherein if the nucleic acid sequence is as defined in SEQ ID No. 1 saidplant is not rice.

In another aspect, the invention relates to a method for regulating pHhomeostasis which may comprise introducing and expressing a nucleic acidconstruct which may comprise a nucleic acid sequence which may compriseSEQ ID No. 1, a functional variant, part or homolog thereof operablylinked to a regulatory sequence in a plant.

In another aspect, the invention relates to a method for reducingacidification in a plant which may comprise introducing and expressing anucleic acid construct which may comprise a nucleic acid sequence whichmay comprise SEQ ID No. 1, a functional variant, part or homolog thereofoperably linked to a regulatory sequence in a plant.

In another aspect, the invention relates to a method for alteringnitrate transport and pH homeostasis in a plant which may compriseintroducing and expressing a nucleic acid construct which may comprise anucleic acid sequence which may comprise SEQ ID No. 1, a functionalvariant, part or homolog thereof operably linked to a regulatorysequence in a plant wherein said nucleic acid comprises a mutation inthe pH sensing motif VYEAIHKI (SEQ ID No. 16).

In another aspect, the invention relates to a use of a nucleic acidwhich may comprise SEQ ID No. 1, a functional variant, part or homologthereof which may comprise the pH sensing motif VYEAIHKI (SEQ ID No. 16)in regulating pH, altering nitrate transport and pH homeostasis in aplant.

In a further aspect, the invention relates to a method for increasingone or more of growth, yield, nitrogen use efficiency, nitrogentransport, nitrogen stress tolerance, pathogen resistance and/ornitrogen acquisition of a plant which may comprise introducing andexpressing a nucleic acid construct which may comprise a nucleic acidsequence as defined in SEQ ID No. 1, a functional variant, part orhomolog thereof operably linked to a regulatory sequence into a plantwherein said regulatory sequence is a constitutive promoter or a phloemspecific promoter and wherein said plant does not overexpress a nucleicacid sequence which may comprise SEQ ID No. 2.

In another aspect, the invention relates to a method for making atransgenic plant having increased growth, yield, nitrogen transport,nitrogen acquisition, nitrogen stress tolerance and/or nitrogen useefficiency which may comprise

a) introducing and expressing in a plant or plant cell a nucleic acidconstruct which may comprise a nucleic acid sequence as defined in SEQID No. 1, a functional variant, part or homolog thereof operably linkedto a regulatory sequence wherein said regulatory sequence is aconstitutive promoter or a phloem specific promoter and wherein saidplant does not overexpress a nucleic acid sequence which may compriseSEQ ID No. 2.

In another aspect, the invention relates to a transgenic plantexpressing a nucleic acid construct which may comprise a nucleic acidsequence as defined in SEQ ID No. 1, a functional variant, part orhomolog thereof operably linked to a regulatory sequence into a plantwherein said regulatory sequence is a constitutive promoter or a phloemspecific promoter and wherein said plant does not overexpress a nucleicacid sequence SEQ ID No. 2.

In another aspect, the invention relates to a transgenic plantexpressing a nucleic acid construct which may comprise a nucleic acidsequence as defined in SEQ ID No. 1, a functional variant, part orhomolog thereof operably linked to a phloem specific promoter andrelated methods.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

The invention is further described in the following non-limitingfigures.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1A-E. The Nipponbare phenotype of OsNRT2.3a and OsNRT2.3bover-expression plants. (a) The T2 rice plants in paddy soil atvegetative (60 days). (b) Reproductive stages (120 days). (c) RT-PCRwith the specific primers. (d) Western blot with mono-antibody toidentify protein expression. (e) The RNA in situ hybridization in WT andb-S6 with negative probe control, p: phloem, x: xylem; e, epidermalcells; m; mesophyll cells. Cross sections are the 5-6 cm leaf sectionfrom the tip of first leaf of plants in (a). Scale bar=10 μm.

FIG. 2A-F. The field experiments of T2 OsNRT2.3b over-expression lines.(a) The growth of OsNRT2.3b over-expression lines b-U1, b-U2, b-S2 andb-S6 at different N fertilizer application rates (May-October 2010, thephotographs were taken on 16 Sep. 2010) at Changxing experiment station,Zhejiang University. The N application was shown in the left corner ofeach picture with the Chinese label given in the middle of the field.(b) The plant grain yield for “a” conditions. (c) The NUE at “a”conditions. (d) Large scale experiment, 1280 seedlings of each typetransferred into paddy soil. (e) Grain yield at “d” condition. (f) TheNUE for “d” conditions. NUE: nitrogen use efficiency=g-grainyield/g-applied fertilizer N. Values are mean±S.E (n=3). * was abovebars indicating significant level (*p<0.05) between the transgenic linesand WT at the same N fertilizer application rate estimated by ANOVA.

FIG. 3A-E. The effects of OsNRT2.3b over-expression on the influx of¹⁵NO₃ ⁻ and ¹⁵NH₄ ⁺ by root, xylem NO₃ ⁻ and NH₄ ⁺, xylem pH, phloem pHacidification at 2.5 mM NO₃ ⁻ or NH₄ ⁺ condition. (a) The ¹⁵N influxrate at nitrate or ammonium. (b) xylem NO₃ ⁻ and NH₄ ⁺ at nitrate orammonium for 24 h. (c) xylem sap pH at nitrate or ammonium. (d) phloempH acidification in nitrate or ammonium, phloem sap was collected byEDTA-Na₂ ¹⁶. (e) Phloem pH acidification in nitrate or ammonium, phloemsap was collected by insects. Values are mean ±S.E (n=5). * was abovebars indicating significant level (*p<0.05) between the transgenic linesand WT at the same treatment estimated by ANOVA. Bars from left to rightin a-d: WT, b-U1, b-U2, b-S2, b-S6

FIG. 4A-C. The effects of OsNRT2.3b over-expression on the influx ofdifferent forms of N at pH 4 and 6. Bars from left to right: WT, b-U1,b-U2, b-S2, b-S6. (a) The ¹⁵N influx in NH₄ ¹⁵NO₃ supply. (b) The ¹⁵Ninflux in ¹⁵NH₄NO₃ supply. (c) The ¹⁵N influx in ¹⁵NH₄ ¹⁵NO₃ supply.Values are mean±S.E (n=5). a, b, c letters were above bars indicatingsignificant difference (p<0.05) between the transgenic lines and WT atthe same treatment estimated by ANOVA.

FIG. 5A-D. The functional analysis of OsNRT2.3b in Xenopus ooctyes. (a)A double barreled pH electrode recording of cytosolic pH from anOsNRT2.3b injection oocyte, treated with 1 mM nitrate (shaded bar) andpH 8.0 saline (grey bar) washing. (b) The membrane potential to 1 mMnitrate (shaded bar) for an oocyte expressing H167R mutant of OsNRT2.3b.(c)¹⁵N-nitrate uptake by oocytes injected with water, OsNRT2.3b mRNAsand its H167R mutant. (d)¹⁵N-nitrate uptake by oocytes injected withwater and OsNRT2.3b mRNAs at different external pH for over-night.Values are mean±S.E (n=15). Cells were tested by electrophysiology to bealive after incubation experiment. * was above bars indicatingsignificant level (*p<0.05) estimated by ANOVA.

FIG. 6A-C. Plant fresh weight (A) and root length (B) tissue nitrateaccumulation (C) data for three Arabidopsis lines over-expressingOsNRT2.3b compared with wild type (wt). Bars from left to right in (C):23b.1, 23b.2, 23b.3, WT

FIG. 7. Comparison of tobacco plants overexpressing OsNRT2.3b and WTplants: phenotype analysis. Growth differences of T1 OsNRT2.3bover-expression lines in sand-filled pots. WT: Nicotiana tabacumcultivar 89, T1 generation grown for 2 months in a complete Hoaglandnutrient solution with 10 mM nitrate supply.

FIG. 8A-B. Comparison of tobacco plants overexpressing OsNRT2.3b and WTplants: expression analysis. A) Southern blot of OsNRT2.3boverexpression lines Kpn I, HindIII digested tobacco DNA of T1generation and Hyb probe was used for hybridization Ld: marker, P:positive control, b-20 is a negative control. B) RT-PCR of OsNRT2.3bover-expression lines cDNA of T1 generation and OsNRT2.3b specificprimer was used for the PCR.

FIG. 9. Biomass and NUE of tobacco overexpressing OsNRT2.3b lines grownin sand-filled pots WT: Nicotiana tabacum cultivar 89, T1 generationgrown for 2 months in a complete Hoagland nutrient solution with 10 mMnitrate supply. NUE=biomass/total N application).

FIG. 10. The gene structure of OsNRT2.3a/b (SEQ ID No. 2 and 1, peptideOsNRT2.3a/b are SEQ ID No. 3 and 4). Analysis of the OsNRT2.3 genomicDNA sequence predicts an intron for OsNRT2.3b located between +190 bp to+280 bp from the ATG for translation initiation. For OsNRT2.3a the5′-UTR is 42 bp and 249 bp 3′-UTR and for OsNRT2.3b the 5′-UTR is 223 bpand 316 bp 3′-UTR. F means the specific forward primer for OsNRT2.3b andR is the reward primer for OsNRT2.3b.

FIG. 11A-F. T2 OsNRT2.3b over-expression plants in the Nipponbarecultivar background in Hainan. a: The T2 Nipponbare transgenic plantswere grown in Ledong Experimental Station of Nanjing AgriculturalUniversity, Hainan Province (December 2009-April 2010). The soilnutrient status before fertilizer addition was total nitrogen (N)1.0±0.2 mg/g, total phosphorus (P) 0.4±0.1 mg/g, total potassium (K)39.5±2.3 mg/g, 0.5 mM NaHCO3 extractable P (Olsen P) 23.1±4.1 mg/kg,soil pH 4.4±0.5 (sampling number was 6). The date for this picture was28 Feb. 2010 and plants were grown for 75 days from germination at 75 kgN/ha N condition. All the planting and fertilizing information wasdescribed as FIG. SF7; b: plant panicle; c: panicle length; d, e:numbers of primary and second rachis; f: grain yield. Values aremean±S.E (n=10), * indicates significance of difference between WT andover-expression plants at 5% levels with One-way ANOVA analysis. Barsfrom left to right: WT, a-U1, a-U2, b-U-1, b-U2, b-S2, b-S6.

FIG. 12A-F. The phenotype of OsNRT2.3b over-expression lines in the WYJ7cultivar background. a: Pot experiment done in Nanjing 2010. The T1lines of 396-2, 369-1, 366-1 and 342-1 were over-expressed withOsNRT2.3b in comparison to its wild type (WT: WYJ7). Seeds weregerminated at 20th May and the picture was taken on 20th October beforeharvest; b: Southern blot of T1 seedlings. Then the 396-2, 369-1, 366-1and 342-1 lines were renamed as 396, 369, 366 and 342 for the T2 fieldexperiments; c: RT-PCR with primers, 26 cycles were set for this PCR. d:T2 field experiments at the Experimental Station of Zhejiang University(May 2011-October 2011) with two application levels as 110 and 220 kgN/ha. Seeds were put to germinate on 5 May 2011, then 100 seedlings weretransferred to the paddy field as 5 rows ×20 plants on 5th June andarranged randomly. Fertilizers were applied as in FIG. 2. The picturewas taken on 10th October before harvest. The soil nutrient statusbefore fertilizer addition was: total nitrogen (N) 1.68±0.21 mg/g, totalphosphorus (P) 0.48±0.18 mg/g, total potassium (K) 46.47±2.85 mg/g, 0.5mM NaHCO3-extractable P 38±2.1 mg/kg, soil pH 6.43±0.28 (n=6); e and f:The grain yield and NUE. Values are mean±S.E (n=3), * indicatessignificance of difference between WT and over-expression plants at 5%levels with One-way ANOVA analysis. Bars from left to right: WYT, 396,369, 366, 342

FIG. 13A-D. The effect of OsNRT2.3b over-expression in the YF47 cultivarbackground. a: Plant growth performance of YF47 (wild type) and thetransgenic plant with over-expression of NRT2.3b (YF/NRT2.3b(O)) infield trails at Hainan Experiment Station of Zhejiang University(December 2011-April 2012). Seeds were put to germinate on 10^(th)December and the photograph was taken on 1^(st) April, 15 days beforethe harvest; b: RT-PCR analysis of the transcript levels of NRT2.3b inYF47 (wild type) and the transgenic plants. c: Southern blot analysis ofthe transgenic plant; d: The grain yield per plant of YF47 and thetransgenic plants. Values are mean±5.E (n=50). The soil nutrient statusbefore fertilizer addition was: total nitrogen (N) 1.5±0.2 mg/g, totalphosphorus (P) 0.3±0.1 mg/g, total potassium (K) 3.5±0.3 mg/g, 0.5 mMNaHCO₃-extractable P 24.1±4.7 mg/kg, soil pH 6.45±0.47 (n=9).

FIG. 14A-B. The T5 phenotype of OsNRT2.3b over-expression lines in theNipponbare cultivar background. a: The T5 Nipponbare transgenic plantsb-S2 and b-S6 were grown in Ledong Experimental Station of NanjingAgricultural University, Hainan Province (December 2011-April 2012), 300seeds were put to germinate on 10^(th) Dec. 200 seedlings weretransferred to the paddy field on 5^(th) January. The picture was takenon the 13^(th) April before harvest. The experimental plot size was 20m×25 m, 60 kg P/ha and 110 kg K/ha fertilizer was applied to the paddybefore transferring the rice seedlings. Two N fertilizer levels wereused 110 and 220 kg N/ha to the paddy. The first N fertilizer wasapplied as 20% of total N treatment before transplanting on 28^(th)December Second application at 40% of total was made at 12^(th) January.The final application was made at the 20^(th) January b: grain yield.

FIG. 15. The F2 generation phenotype of Nipponbare (♀)×b-S6 T5(♂).

FIG. 16A-I. The phenotype difference between OsNRT2.3b over-expressionplants and WT in pot experiments at late growth stage. This potexperiment was conducted as described in Table 1 and the growth wasrecorded at 76 days (a); 84 days (b); 88 days (c); 98 days (d); 120 days(e) and 140 days after transplant (f). The grain yield (g) total N (h)and NU_(t)E=grain weight/total N (i) of WT, b-S2 and b-S6 were measuredat 120 and 140 days, separately. Values are mean±S.E (n=10), * indicatessignificance of difference between WT and over-expression plants at 5%levels with One-way ANOVA analysis. The pictures were taken only with WTand b-S6 because two plants were easily distinguished compared with allthree plants in picture. Therefore black cloth was used as backgroundand separated WT and b-S6 from b-S2, which was behind of the cloth inpot.

FIG. 17A-B. The method for phloem sap sampling from the Brown PlantHopper (Nilapavata lugens). Rice seedlings were grown hydroponically in1.25 mM NH₄NO₃ for 8 weeks and then transferred to N treatments (N: 2.5mM NO₃ ⁻; A: 2.5 mM NH₄ ⁻). Each plant was placed in a 250 ml flask ofIRRI nutrient solution with six plants kept in the insect cage at 26° C.and a 16 h light period. Seven to ten brown plant hopper adults weretransferred on to each plant at the beginning of the N treatments. Ricephloem honey dew secreted by the insects was collected at 24 h, 48 h ofthe N treatments. Phloem sap pH was measured using a pH selectivemicroelectrode²²; a: phloem pH in nitrate; b: phloem pH in ammonium.Values are mean±S.E (n =10), * indicates significance of differencebetween WT and b-S6 at 5% levels with One-way ANOVA analysis.

FIG. 18A-C. The root apoplastic pH in the line b-S6 of OsNRT2.3bover-expression and WT after 72 h N treatment. Rice seedlings were grownin full nutrient solution containing 1.25 mM NH₄NO₃ for 4 weeks and thentransferred to N treatments (N: 2.5 mM NO₃ ⁻; A: 2.5 mM NH₄ ⁻) for 72 h.a: the apoplastic pH of rice roots. After 72 h N treatment, the plantroot was washed by dipping into 0.2 mM CaSO₄ for one minute beforeplacement on the agar¹⁷. An intact plant was placed on agar (0.9 g/l,containing the pH indicator (0.03 g/L bromocresol purple¹⁷). The initialpH was 5.2-5.3 from 11:00-11:30 am, and roots were kept in darknesscovered with a moist paper tissue and under a 0.5×12×12 cm³ Plexiglasplate and picture was taken after 2-4 h in contact with the pH indicatoragar; b: Agar profile showing apoplastic pH after removing the roots; c:the longer term pH change of the hydroponic growth medium during the Ntreatments.

FIG. 19. The total leaf P and Fe in T2 Nipponbare rice over-expressingOsNRT2.3b growing in 1.25 mM NH₄NO₃ hydroponic culture. The total P andFe was measured by ICP analysis. The 0.05 g dried crushed plant materialpowder was digested with 5 ml of 98% H₂SO₄ and 3 ml of 30% hydrogenperoxide. After cooling, the digested sample was diluted to 100 ml withdistilled water. The ion concentrations in the solution were measuredusing the ICP-OES (Perkin Elmer Optima 2000 DV). Values are mean±S.E(n=4), * indicates significance of difference between WT andover-expression plants at 5% levels with One-way ANOVA analysis. Barsfrom left to right: WT, b-U1, b-U2, b-S2, b-S6.

FIG. 20A-C. The total photosynthesis, intercellular CO₂ concentrationand photorespiration in plants over-expressing OsNRT2.3b compared withWT. The net photosynthesis, intercellular CO₂ concentration andphotorespiration were measured using a Li-Cor 6400 infrared gas analyzeras described before⁴¹. a: total photosynthesis was calculated by netphotosynthesis times the measured leaf area; b: intercellular CO₂concentration; c: The net dark respiration (R_(n)) was reached duringCO₂ PIB recording at stable recording stage from 100 to 200 secondsafter shutting off lights, according to Supplemental FIG. 4 of Kebeishet al., 2007²². Values are mean±S.E (n=4), * indicates significance ofdifference between WT and over-expression plants at 5% levels withOne-way ANOVA analysis.

FIG. 21A-D. The over-expression of OsNRT2.3b H167R mutant in Nipponbare.a: F1 generation plants of over-expression of OsNRT2.3b H167R mutantlines OvH1, OvH2 and WT in pot experiment (May.2012-September 2012) allthe planting systems were the same as in Table 1. The photograph wastaken on 10^(th) September; b: grain weight. Values are mean±S.E (n=60);c: RT-PCR with the same primers for OsNRT2.3b, which covers the mutatedsite; d: southern blot.

FIG. 22. ¹⁵N—NH₄ ⁺ uptake by oocytes injected with water or OsNRT2.3bmRNA. 0.5 mM ¹⁵N—NH₄Cl (atom % ¹⁵N 98%) was added into ND96 solution andthe oocytes were incubated overnight (16 h). Values are mean±S.E (n=15).

FIG. 23A-D. The field design for the experiments shown in FIG. 2a andFIG. 2d . T2 field experiments were conducted in Changxing experimentstation of Zhejiang University. For FIG. 2a the plants were transferredto the right blocks with four N application levels: no nitrogen, 75 kgN/ha, 150 kg N/ha and 300 kg N/ha; For FIG. 2d , plants were transferredto the left blocks with 75 kg N/ha supply. Each experimental block sizewas 20 m×30 m and 60 kg P/ha and 110 kg K/ha fertilizer was applied tothe paddy before transferring the rice seedlings; b: the N treatments ineach block; c: the plant arrangement in FIG. 2a with the same row andplant spaces as d; d: the plant arrangement in FIG. 2d . All fieldexperiments were conducted with three replications randomly arranged.

FIG. 24A-B. Table showing putative NRT2 nitrate transporters which havethe pH-sensing motif that was identified in OsNRT2.3b.

-   -   *best candidate for OsNRT2.3 orthologs    -   Databases for searches: Blast sequence searches from phytozome 9        http://www.phytozome.net/

Wheat database:

-   -   http://www.cerealsdb.uk.net/CerealsDB/Documents/DOC_search_reads.php    -   Barley database: http://webblast.ipk-gatersleben.de/barley/    -   Membrane protein anion exchanger motifs (containing pH sensor)        identified using    -   http://www.bioinf.manchester.ac.uk/cgi-bin/dbbrowser/fingerPRINTScan/FPScan_fam.cgi

FIG. 25. Overexpression OsNRT2.3b will enhance the phloem pH balancing.WT, wild type rice plant; b-S6, OsNRT2.3b over expression line; H167R,OsNRT2.3b H167R over expression line. The phloem pH was measured by pHselective electrode. Phloem sap was harvested by the Brown Plant Hopper(Nilapavata lugens) method. Rice seedlings were grown hydroponically in1.25 mM NH₄NO₃ for 8 weeks and then transferred to N treatments (N: 2.5mM NO₃ ⁻; A: 2.5 mM NH₄ ⁺). Each plant was placed in a 250 ml flask ofIRRI nutrient solution with six plants kept in the insect cage at 26 Cand a 16 h light period. Seven to ten brown plant hopper adults weretransferred on to each plant at the beginning of the N treatments. Ricephloem honey dew secreted by the insects was collected at 24 h after theN treatments began. The results showed that WT and H167R line of phloempH were same pattern at different N form however b-S6 was more near to 7at nitrate treatment, more near neutral in both N conditions.

FIG. 26. Survival data for transgenic rice.

FIG. 27. The biomass of wheat OsNRT2.3b transgenic lines.

FIG. 28. The growth of wheat OsNRT2.3b transgenic lines in low and highN application.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the followingpassages, different aspects of the invention are defined in more detail.Each aspect so defined may be combined with any other aspect or aspectsunless clearly indicated to the contrary. In particular, any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry and recombinant DNAtechnology, bioinformatics which are within the skill of the art. Suchtechniques are explained fully in the literature.

As used herein, the words “nucleic acid”, “nucleic acid sequence”,“nucleotide”, “nucleic acid molecule” or “polynucleotide” are intendedto include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules(e.g., mRNA), natural occurring, mutated, synthetic DNA or RNAmolecules, and analogs of the DNA or RNA generated using nucleotideanalogs. It can be single-stranded or double-stranded. Such nucleicacids or polynucleotides include, but are not limited to, codingsequences of structural genes, anti-sense sequences, and non-codingregulatory sequences that do not encode mRNAs or protein products. Theseterms also encompass a gene. The term “gene” or “gene sequence” is usedbroadly to refer to a DNA nucleic acid associated with a biologicalfunction. Thus, genes may include introns and exons as in the genomicsequence, or may comprise only a coding sequence as in cDNAs, and/or mayinclude cDNAs in combination with regulatory sequences.

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to amino acids in a polymeric form of any length, linkedtogether by peptide bonds.

For the purposes of the invention, “transgenic”, “transgene” or“recombinant” means with regard to, for example, a nucleic acidsequence, an expression cassette, gene construct or a vector which maycomprise the nucleic acid sequence or an organism transformed with thenucleic acid sequences, expression cassettes or vectors according to theinvention, all those constructions brought about by recombinant methodsin which either

-   -   (a) the nucleic acid sequences encoding proteins useful in the        methods of the invention, or    -   (b) genetic control sequence(s) which is operably linked with        the nucleic acid sequence according to the invention, for        example a promoter, or    -   (c) a) and b)

are not located in their natural genetic environment or have beenmodified by recombinant methods, it being possible for the modificationto take the form of, for example, a substitution, addition, deletion,inversion or insertion of one or more nucleotide residues. The naturalgenetic environment is understood as meaning the natural genomic orchromosomal locus in the original plant or the presence in a genomiclibrary. In the case of a genomic library, the natural geneticenvironment of the nucleic acid sequence is preferably retained, atleast in part. The environment flanks the nucleic acid sequence at leaston one side and has a sequence length of at least 50 bp, preferably atleast 500 bp, especially preferably at least 1000 bp, most preferably atleast 5000 bp. A naturally occurring expression cassette—for example thenaturally occurring combination of the natural promoter of the nucleicacid sequences with the corresponding nucleic acid sequence encoding apolypeptide useful in the methods of the present invention, as definedabove—becomes a transgenic expression cassette when this expressioncassette is modified by non-natural, synthetic (“artificial”) methodssuch as, for example, mutagenic treatment. Suitable methods aredescribed, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815 bothincorporated by reference.

A transgenic plant for the purposes of the invention is thus understoodas meaning, as above, that the nucleic acids used in the method of theinvention are not at their natural locus in the genome of said plant, itbeing possible for the nucleic acids to be expressed homologously orheterologously. However, as mentioned, transgenic also means that, whilethe nucleic acids according to the different embodiments of theinvention are at their natural position in the genome of a plant, thesequence has been modified with regard to the natural sequence, and/orthat the regulatory sequences of the natural sequences have beenmodified. Transgenic is preferably understood as meaning the expressionof the nucleic acids according to the invention at an unnatural locus inthe genome, i.e. homologous or, preferably, heterologous expression ofthe nucleic acids takes place.

The aspects of the invention involve recombination DNA technology andexclude embodiments that are solely based on generating plants bytraditional breeding methods.

The OsNRT2.3b peptide expressed according to the aspects of theinvention is shown in SEQ ID No. 3. According to the aspects of theinvention, nucleic acid sequence SEQ ID No. 1 (OsNRT2.3b) encodespolypeptide SEQ ID No. 3 (OsNRT2.3b). Nucleic acid sequence SEQ ID No. 2(OsNRT2.3a) encodes polypeptide SEQ ID No. 4 (OsNRT2.3a). Constructsthat may comprise SEQ ID No. 67 which corresponds to accession No.AK072215 of OsNRT2.3b according to all embodiments and aspects of theinvention. When referring to a nucleic acid encoding to OsNRT2.3a, thisalso refers to accession No. AK0109776 of OsNRT2.3a as shown in SEQ IDNo. 68 according to all embodiments and aspects of the invention.

The inventors have demonstrated that over-expressing OsNRT2.3b indifferent rice cultivars increased grain yield by up to 40% and improvedNUE under both low and high N inputs in extensive field trials.Photorespiratory gene expression was decreased in rice over-expressingOsNRT2.3b showing that improved photosynthetic efficiency is a componentof the enhanced yield phenotype. Interestingly, the OsNRT2.3bover-expression lines, which were confirmed at both transcript andprotein levels (FIG. 1c, d ), showed more growth compared with wild type(WT) (FIG. 1a, b , FIG. 11). The biomass and panicle size ofover-expression lines was greater than WT (FIG. 11, Table 2-3). Theprimary and second rachis size was increased, therefore the total numberof seeds per panicle was greater than WT (FIG. 11, Table 2). Bycontrast, the OsNRT2.3a over-expression plants did not show visibledifference from WT even though OsNRT2.3a mRNA and protein was increasedin the transformed lines (FIG. 1c, d , FIG. 11).

Over-expressing OsNRT2.3b also improved pH homeostasis that resulted inincreased total N uptake, shoot P and Fe accumulation. These resultsdemonstrate that linking N uptake to pH homeostasis and photosynthesisis a key consideration for improving NUE and yield.

Thus, the inventors have demonstrated that OsNRT2.3b, but not OsNRT2.3a,can be used to improve growth, yield and nitrogen use efficiency andother traits when expressed in a plant. Accordingly, in some aspects,the invention relates to methods, uses and plants expressing a nucleicacid sequence which may comprise a nucleic acid as defined as defined inSEQ ID No. 1 (OsNRT2.3b), a functional variant, part or homolog thereof,but wherein said plant does not expressing a nucleic acid sequence whichmay comprise a nucleic acid as defined as defined in SEQ ID No. 2(OsNRT2.3a). In particular, the invention therefore relates to methodsfor increasing growth, yield, nitrogen transport, pathogen resistance,NUE and/or nitrogen acquisition which may comprise introducing andexpressing a nucleic acid construct which may comprise a nucleic acidsequence as defined in SEQ ID No. 1 (OsNRT2.3b) operably linked to aregulatory sequence into a plant wherein said regulatory sequence is aconstitutive promoter or a phloem specific promoter and wherein saidplant does not overexpress a nucleic acid sequence which may compriseSEQ ID No. 2 (OsNRT2.3a).

The invention has a further aspect. As mentioned above, rice differsfrom all other major crop in its nitrogen metabolism. Surprisingly, theinventors have shown that expression of OsNRT2.3b from rice, a plantthat is, in contrast to all other major crop plants, capable of growingvigorously on NH₄, is active when expressed in other plant species thatuse NO₃ ⁻ as their nitrogen source. Moreover, expression of OsNRT2.3b inother plants leads to a beneficial phenotype that shows improved growth,yield and nitrogen use efficiency, not only in rice, but also otherplants. Thus, OsNRT2.3b from rice can be used in methods for improvinggrowth, yield, pathogen resistance and nitrogen use efficiency in plantsaccording to the invention. For example, overexpression of OsNRT2.3b intobacco or in wheat increases biomass as shown in the examples.

Thus, the invention also relates to a method for increasing growth,yield, NUE, nitrogen acquisition, nitrogen stress tolerance, pathogenresistance and/or nitrogen transport of a plant which may compriseintroducing and expressing a nucleic acid sequence which may comprise anucleic acid as defined as defined in SEQ ID No. 1, a functionalvariant, part or homolog thereof operably linked to a regulatorysequence in a plant wherein if the nucleic acid sequence is as definedin SEQ ID No. 1 or a functional variant, part thereof said plant is notrice. In a preferred embodiment, the invention relates to a method forincreasing growth, yield, NUE, nitrogen acquisition, nitrogen stresstolerance, pathogen resistance and/or nitrogen transport of a plant thatis not rice which may comprise introducing and expressing a nucleic acidsequence which may comprise a nucleic acid as defined as defined in SEQID No. 1, a functional variant or part thereof operably linked to aregulatory sequence in said plant.

In another aspect, the invention relates to a method for increasinggrowth, yield, NUE, nitrogen acquisition, pathogen resistance, nitrogenstress tolerance and/or nitrogen transport of a plant which may compriseintroducing and expressing a nucleic acid sequence which may comprise oras defined in SEQ ID No. 1, a functional variant, part or homologthereof operably linked to a regulatory sequence in a plant wherein saidplant is not rice.

Thus, in one aspect, the invention relates to a method for increasinggrowth of a plant which may comprise introducing and expressing anucleic acid sequence which may comprise SEQ ID No. 1, a functionalvariant, part or homolog thereof operably linked to a regulatorysequence in a plant wherein if the nucleic acid sequence is as definedin SEQ ID No. 1 said plant is not rice.

In yet another aspect, the invention relates to a method for increasingyield of a plant which may comprise introducing and expressing a nucleicacid sequence which may comprise SEQ ID No. 1, a functional variant,part or homolog thereof operably linked to a regulatory sequence in aplant wherein if the nucleic acid sequence is as defined in SEQ ID No. 1said plant is not rice.

The term “yield” includes one or more of the following non-limitativelist of features: early flowering time, biomass (vegetative biomass(root and/or shoot biomass) or seed/grain biomass), seed/grain yield,seed/grain viability and germination efficiency, seed/grain size, starchcontent of grain, early vigour, greenness index, increased growth rate,delayed senescence of green tissue. The term “yield” in general means ameasurable produce of economic value, typically related to a specifiedcrop, to an area, and to a period of time. Individual plant partsdirectly contribute to yield based on their number, size and/or weight.The actual yield is the yield per square meter for a crop and year,which is determined by dividing total production (includes bothharvested and appraised production) by planted square meters.

Thus, according to the invention, yield may comprise one or more of andcan be measured by assessing one or more of: increased seed yield perplant, increased seed filling rate, increased number of filled seeds,increased harvest index, increased viability/germination efficiency,increased number or size of seeds/capsules/pods/grain, increased growthor increased branching, for example inflorescences with more branches,increased biomass or grain fill. Preferably, increased yield maycomprise an increased number of grain/seed/capsules/pods, increasedbiomass, increased growth, increased number of floral organs and/orfloral increased branching. Yield is increased relative to a controlplant.

For example, the yield is increased by 2%, 3%, 4%, 5%-50% or morecompared to a control plant, for example by at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% or 50%.

In another aspect, the invention relates to a method for increasing NUEof a plant which may comprise introducing and expressing a nucleic acidsequence which may comprise SEQ ID No. 1, a functional variant, part orhomolog thereof operably linked to a regulatory sequence in a plantwherein if the nucleic acid sequence is as defined in SEQ ID No. 1 saidplant is not rice. In another aspect, the invention relates to a methodfor increasing NUE of a plant which may comprise introducing andexpressing a nucleic acid sequence which may comprise SEQ ID No. 1, afunctional variant, part or homolog thereof operably linked to aregulatory sequence in a plant wherein said plant is not rice.

In one embodiment, the method improves NUE under high N input. Inanother embodiment, the method improves NUE under low N input.

NUE can be defined as being the yield of grain per unit of available Nin the soil (including the residual N present in the soil and thefertilizer). The overall N use efficiency of plants may comprise bothuptake and utilization efficiencies and can be calculated as UpE.

For example, the NUE is increased by 5%-50% or more compared to acontrol plant, for example by at least 10%, 15%, 20%, 25%, 30%, 35%,40%, 45% or 50%.

In another aspect, the invention relates to a method for increasingnitrogen acquisition of a plant which may comprise introducing andexpressing a nucleic acid sequence which may comprise SEQ ID No. 1, afunctional variant, part or homolog thereof operably linked to aregulatory sequence in a plant wherein if the nucleic acid sequence isas defined in SEQ ID No. 1 said plant is not rice. In another aspect,the invention relates to a method for increasing nitrogen acquisition ofa plant which may comprise introducing and expressing a nucleic acidsequence which may comprise SEQ ID No. 1, a functional variant, part orhomolog thereof operably linked to a regulatory sequence in a plantwherein said plant is not rice.

For example, the nitrogen acquisition is increased by 10%-50% or morecompared to a control plant, for example by at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% or 50%.

In one embodiment of the various methods described herein for increasingNUE, growth, yield, nitrogen acquisition and/or nitrate transport, saidtraits are increased under stress conditions, for example nitrogenstress.

In another aspect, the invention relates to a method for increasingnitrogen stress tolerance of a plant which may comprise introducing andexpressing a nucleic acid sequence which may comprise SEQ ID No. 1, afunctional variant, part or homolog thereof operably linked to aregulatory sequence in a plant wherein if the nucleic acid sequence isas defined in SEQ ID No. 1 said plant is not rice. In another aspect,the invention relates to a method for increasing nitrogen stresstolerance of a plant which may comprise introducing and expressing anucleic acid sequence which may comprise SEQ ID No. 1, a functionalvariant, part or homolog thereof operably linked to a regulatorysequence in a plant wherein said plant is not rice.

In another aspect, the invention relates to a method for increasingnitrogen transport of a plant which may comprise introducing andexpressing a nucleic acid sequence which may comprise SEQ ID No. 1, afunctional variant, part or homolog thereof operably linked to aregulatory sequence in a plant wherein if the nucleic acid sequence isas defined in SEQ ID No. 1 said plant is not rice. In another aspect,the invention relates to a method for increasing nitrogen transport of aplant which may comprise introducing and expressing a nucleic acidsequence which may comprise SEQ ID No. 1, a functional variant, part orhomolog thereof operably linked to a regulatory sequence in a plantwherein said plant is not rice.

In another aspect, the invention relates to a method for increasingpathogen resistance and/or survival of a plant which may compriseintroducing and expressing a nucleic acid sequence which may compriseSEQ ID No. 1, a functional variant, part or homolog thereof operablylinked to a regulatory sequence in a plant wherein if the nucleic acidsequence is as defined in SEQ ID No. 1 said plant is not rice. Inanother aspect, the invention relates to a method for increasingpathogen resistance and/or survival of a plant which may compriseintroducing and expressing a nucleic acid sequence which may compriseSEQ ID No. 1, a functional variant, part or homolog thereof operablylinked to a regulatory sequence in a plant wherein said plant is notrice.

The pathogen can for example be Fusarium wilt. Other pathogens known tothe skilled persons are also within the scope of the invention.

The terms “regulatory element”, “regulatory sequence”, “controlsequence” and “promoter” are all used interchangeably herein and are tobe taken in a broad context to refer to regulatory nucleic acidsequences capable of effecting expression of the sequences to which theyare ligated. The term “promoter” typically refers to a nucleic acidcontrol sequence located upstream from the transcriptional start of agene and which is involved in recognising and binding of RNA polymeraseand other proteins, thereby directing transcription of an operablylinked nucleic acid. Encompassed by the aforementioned terms aretranscriptional regulatory sequences derived from a classical eukaryoticgenomic gene (including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence) andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner.Also included within the term is a transcriptional regulatory sequenceof a classical prokaryotic gene, in which case it may include a −35 boxsequence and/or −10 box transcriptional regulatory sequences. The term“regulatory element” also encompasses a synthetic fusion molecule orderivative that confers, activates or enhances expression of a nucleicacid molecule in a cell, tissue or organ. Furthermore, the term“regulatory element” includes downstream transcription terminatorsequences. A transcription terminator is a section of nucleic acidsequence that marks the end of a gene or operon in genomic DNA duringtranscription. Transcription terminator used in construct to expressplant genes are well known in the art.

In one embodiment, the constructs described herein have a promoter and aterminator sequence.

A “plant promoter” may comprise regulatory elements, which mediate theexpression of a coding sequence segment in plant cells. Accordingly, aplant promoter need not be of plant origin, but may originate fromviruses or micro-organisms, for example from viruses which attack plantcells. The “plant promoter” can also originate from a plant cell, e.g.from the plant which is transformed with the nucleic acid sequence to beexpressed in the inventive process and described herein. This alsoapplies to other “plant” regulatory signals, such as “plant”terminators. The promoters upstream of the nucleotide sequences usefulin the methods of the present invention can be modified by one or morenucleotide substitution(s), insertion(s) and/or deletion(s) withoutinterfering with the functionality or activity of either the promoters,the open reading frame (ORF) or the 3′-regulatory region such asterminators or other 3′ regulatory regions which are located away fromthe ORF. It is furthermore possible that the activity of the promotersis increased by modification of their sequence, or that they arereplaced completely by more active promoters, even promoters fromheterologous organisms. For expression in plants, the nucleic acidmolecule must, as described above, be linked operably to or may comprisea suitable promoter which expresses the gene at the right point in timeand with the required spatial expression pattern. The term “operablylinked” as used herein refers to a functional linkage between thepromoter sequence and the gene of interest, such that the promotersequence is able to initiate transcription of the gene of interest.

The following promoters may be selected according to the aspects of theinvention. This list is not limiting.

A “constitutive promoter” refers to a promoter that is transcriptionallyactive during most, but not necessarily all, phases of growth anddevelopment and under most environmental conditions, in at least onecell, tissue or organ. Examples of constitutive promoters include butare not limited to actin, HMGP, CaMV19S, GOS2, rice cyclophilin, maizeH3 histone, alfalfa H3 histone, 34S FMV, rubisco small subunit, OCS,SAD1, SAD2, nos, V-ATPase, super promoter, G-box proteins and syntheticpromoters.

A “strong promoter” refers to a promoter that leads to increased oroverexpression of the gene. Examples of strong promoters include, butare not limited to, CaMV-35S, CaMV-35Somega, Arabidopsis ubiquitin UBQ1,rice ubiquitin, actin, or Maize alcohol dehydrogenase 1 promoter(Adh-1).

In a preferred embodiment, the promoter is a constitutive promoters thatis a strong promoter and directs overexpression of the gene of interestto which it is operably linked. Preferred promoters are CaMV-35S,CaMV-35Somega and Arabidopsis ubiquitin UBQ1.

The term “increased expression” or “overexpression” as used herein meansany form of expression that is additional to the control, for examplewild-type, expression level.

In one embodiment, the promoter is a phloem-specific promoter.Phloem-specific expression may be important for the function of theOsNRT2.3b, as the vascular tissue is important for pH regulation and ithas recently been shown that nitrate transport in the phloem occurs inplants and may be a significant route for nitrogen delivery to theshoot.

A phloem specific promoter is, for example, from RSS1P, derived from therice sucrose synthase gene (corresponding to SEQ ID No. 5 or afunctional variant or part thereof, see Saha et al). Otherphloem-specific promoters are known in the art.

According to the various aspects of the invention, growth, yield,nitrogen transport, nitrogen acquisition, nitrogen stress tolerance,pathogen resistance and/or nitrogen use efficiency is increased comparedto a control plant. A control plant is a plant which has not beentransformed with a nucleic acid construct which may comprise SEQ ID No.1, a functional variant, part or homolog thereof, preferably a wild typeplant. The control plant is preferably of the same species as thetransgenic plant. Furthermore, the control plant may comprise geneticmodifications, including expression of other transgenes.

The terms “increase”, “improve” or “enhance” as used according to thevarious aspects of the invention are interchangeable. Growth, yield,nitrogen transport, nitrogen acquisition, nitrogen stress toleranceand/or nitrogen use efficiency is increased by about 5-50%, for exampleat least 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, morepreferably 25%, 30%, 35%, 40%, 45% or 50% or more in comparison to acontrol plant. Preferably, growth is measured by measuring hypocotyl orstem length. In one embodiment, yield is increased by at least 40%.

The nucleic acid construct which may comprise SEQ ID No. 1, a functionalvariant, part or homolog thereof may also comprise a selectable markerwhich facilitates the selection of transformants, such as a marker thatconfers resistance to antibiotics, for example kanamycin.

In another aspect, the invention relates to a method for making atransgenic plant having increased yield, growth, nitrogen transport,nitrogen acquisition, nitrogen stress tolerance, pathogen resistanceand/or nitrogen use efficiency which may comprise introducing andexpressing in a plant or plant cell a nucleic acid sequence which maycomprise SEQ ID No. 1, a functional variant, part or homolog thereofoperably linked to a regulatory sequence wherein if the nucleic acidsequence is as defined in SEQ ID No. 1 said plant is not rice. Inanother aspect, the invention relates to a method for making atransgenic plant having increased yield, growth, nitrogen transport,nitrogen acquisition, nitrogen stress tolerance and/or nitrogen useefficiency which may comprise introducing and expressing in a plant orplant cell a nucleic acid sequence which may comprise SEQ ID No. 1, afunctional variant, part or homolog thereof operably linked to aregulatory sequence wherein said plant is not rice.

The method further may comprise regenerating a transgenic plant from theplant or plant cell after step a) wherein the transgenic plant maycomprise in its genome SEQ ID No. 1, a functional variant, part orhomolog thereof operably linked to a regulatory sequence and obtaining aprogeny plant derived from the transgenic plant wherein said progenyplant exhibits increased yield, growth, nitrogen transport, nitrogenacquisition, nitrogen stress tolerance and/or nitrogen use efficiency.

In one embodiment of these methods described above which explicitlyexclude rice, the nucleic acid sequence may comprise or consists of SEQID No. 1 or a functional variant or part thereof.

Thus, according to the various aspects of the invention, SEQ ID No. 1, afunctional variant, part or homolog thereof is introduced into a plantand expressed as a transgene. The nucleic acid sequence is introducedinto said plant through a process called transformation. The term“introduction” or “transformation” as referred to herein encompasses thetransfer of an exogenous polynucleotide into a host cell, irrespectiveof the method used for transfer. Plant tissue capable of subsequentclonal propagation, whether by organogenesis or embryogenesis, may betransformed with a genetic construct of the present invention and awhole plant regenerated there from. The particular tissue chosen willvary depending on the clonal propagation systems available for, and bestsuited to, the particular species being transformed. Exemplary tissuetargets include leaf disks, pollen, embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristem, axillary buds, and root meristems), and inducedmeristem tissue (e.g., cotyledon meristem and hypocotyl meristem). Thepolynucleotide may be transiently or stably introduced into a host celland may be maintained non-integrated, for example, as a plasmid.Alternatively, it may be integrated into the host genome. The resultingtransformed plant cell may then be used to regenerate a transformedplant in a manner known to persons skilled in the art.

The transfer of foreign genes into the genome of a plant is calledtransformation. Transformation of plants is now a routine technique inmany species. Advantageously, any of several transformation methods maybe used to introduce the gene of interest into a suitable ancestor cell.The methods described for the transformation and regeneration of plantsfrom plant tissues or plant cells may be utilized for transient or forstable transformation. Transformation methods include the use ofliposomes, electroporation, chemicals that increase free DNA uptake,injection of the DNA directly into the plant, particle gun bombardment,transformation using viruses or pollen and microprojection. Methods maybe selected from the calcium/polyethylene glycol method for protoplasts,electroporation of protoplasts, microinjection into plant material, DNAor RNA-coated particle bombardment, infection with (non-integrative)viruses and the like. Transgenic plants, including transgenic cropplants, are preferably produced via Agrobacterium tumefaciens mediatedtransformation.

To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above. Following DNAtransfer and regeneration, putatively transformed plants may also beevaluated, for instance using Southern analysis, for the presence of thegene of interest, copy number and/or genomic organisation. Alternativelyor additionally, expression levels of the newly introduced DNA may bemonitored using Northern and/or Western analysis, both techniques beingwell known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques. The generated transformed organisms may take a variety offorms. For example, they may be chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g., all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissues (e.g., in plants, a transformed rootstock graftedto an untransformed scion).

The various aspects of the invention described herein clearly extend toany plant cell or any plant produced, obtained or obtainable by any ofthe methods described herein, and to all plant parts and propagulesthereof unless otherwise specified. For example, in certain aspectsdescribed above, rice is specifically excluded. Thus, the methodsexclude embodiments where a nucleic acid which may comprise or consistof SEQ ID No. 1 or a functional part of variant thereof are is expressedin rice. The present invention extends further to encompass the progenyof a primary transformed or transfected cell, tissue, organ or wholeplant that has been produced by any of the aforementioned methods, theonly requirement being that progeny exhibit the same genotypic and/orphenotypic characteristic(s) as those produced by the parent in themethods according to the invention.

The plant of the various aspects of the invention is characterised inthat it shows increased growth, yield, nitrogen transport, nitrogenacquisition, nitrogen stress tolerance and/or nitrogen use efficiency.

The invention also extends to harvestable parts of a plant of theinvention as described above such as, but not limited to seeds, leaves,fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The inventionfurthermore relates to products derived, preferably directly derived,from a harvestable part of such a plant, such as dry pellets or powders,oil, fat and fatty acids, starch or proteins.

The invention also relates to the use of a sequence which may compriseSEQ ID No. 1, a functional variant, part or homolog thereof inincreasing growth, yield, NUE, nitrogen acquisition, nitrogen stresstolerance, pathogen resistance and/or nitrogen transport of a plantwherein if the SEQ may comprise SEQ ID No. 1, said plant is not rice.Further, the invention also relates to the use of a sequence which maycomprise SEQ ID No. 1, a functional variant, part or homolog thereof inincreasing growth, yield, NUE, nitrogen acquisition, nitrogen stresstolerance and/or nitrogen transport of a plant wherein said plant is notrice.

The invention also relates to a nucleic acid construct which maycomprise nucleic acid sequence SEQ ID No. 1, a functional variant, partor homolog operably linked to a phloem specific promoter, for example anucleic acid which may comprise SEQ ID No. 5. Further provided is theuse of the construct in the methods described herein.

Also provided is an isolated cell, preferably a plant cell or anAgrobacterium tumefaciens cell, expressing a nucleic acid constructwhich may comprise nucleic acid sequence SEQ ID No. 1, a functionalvariant, part or homolog operably linked to a phloem specific promoter.In another aspect, the invention relates to an isolated cell, preferablya plant cell or an Agrobacterium tumefaciens cell expressing a nucleicacid construct which may comprise nucleic acid sequence SEQ ID No. 1, afunctional variant, part or homolog operably linked to a constitutivepromoter. Furthermore, the invention also relates to a culture mediumwhich may comprise an isolated plant cell or an Agrobacteriumtumefaciens cell expressing a nucleic acid construct of the invention.

Unless rice is specifically disclaimed, the transgenic plant accordingto the various aspects of the invention described herein may be anymonocot or a dicot plant provided for the embodiments described herein.

A dicot plant may be selected from the families including, but notlimited to Asteraceae, Brassicaceae (eg Brassica napus), Chenopodiaceae,Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae,Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. Forexample, the plant may be selected from lettuce, sunflower, Arabidopsis,broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam,capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean,soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots,pears, peach, grape vine or citrus species. In one embodiment, the plantis oilseed rape.

Also included are biofuel and bioenergy crops such as rape/canola, sugarcane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin andwillow, poplar, poplar hybrids, Miscanthus or gymnosperms, such asloblolly pine. Also included are crops for silage (maize), grazing orfodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax),building materials (e.g. pine, oak), pulping (e.g. poplar), feederstocks for the chemical industry (e.g. high erucic acid oil seed rape,linseed) and for amenity purposes (e.g. turf grasses for golf courses),ornamentals for public and private gardens (e.g. snapdragon, petunia,roses, geranium, Nicotiana sp.) and plants and cut flowers for the home(African violets, Begonias, chrysanthemums, geraniums, Coleus spiderplants, Dracaena, rubber plant).

A monocot plant may, for example, be selected from the familiesArecaceae, Amaryllidaceae or Poaceae. For example, the plant may be acereal crop, such as wheat, barley, maize, oat, sorghum, rye, millet,buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species,or a crop such as onion, leek, yam or banana. In one embodiment of themethods and plants described above, the plant is not rice.

Preferably, the plant is a crop plant. By crop plant is meant any plantwhich is grown on a commercial scale for human or animal consumption oruse.

Most preferred plants are maize, wheat, oilseed rape, sorghum, soybean,potato, tobacco tomato, tobacco, grape, barley, pea, bean, field bean,lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetablebrassicas or poplar.

In one embodiment, the plant is wheat. In one embodiment, the plant istobacco. Preferably, the promoter is a phloem specific promoter asdescribed herein.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, fruit, shoots,stems, leaves, roots (including tubers), flowers, and tissues andorgans, wherein each of the aforementioned may comprise the gene/nucleicacid of interest. The term “plant” also encompasses plant cells,suspension cultures, callus tissue, embryos, meristematic regions,gametophytes, sporophytes, pollen and microspores, again wherein each ofthe aforementioned may comprise the gene/nucleic acid of interest.

Plants or parts thereof obtained or obtainable by the method for makinga transgenic plant as described above are also within the scope of theinvention.

In another aspect, the invention relates to a transgenic plantexpressing a nucleic acid sequence which may comprise SEQ ID No. 1, afunctional variant, part or homolog thereof operably linked to aregulatory sequence into a plant wherein if the nucleic acid sequence isas defined in SEQ ID No. 1 said plant is not rice. Thus, this aspect ofthe invention excludes transgenic rice expressing a nucleic acid whichmay comprise or consist of SEQ ID No. 1. In one embodiment, other plantsthat are capable of growing on NH₄ as the sole nitrogen source are alsoexcluded.

In another aspect, the invention relates to a transgenic plantexpressing a nucleic acid sequence which may comprise SEQ ID No. 1, afunctional variant, part or homolog thereof operably linked to a phloemspecific promoter in a plant. The plant may be any monocot or dicotplant, including rice. In one embodiment, said plant is not rice

In another aspect, the invention relates to a transgenic plantexpressing a nucleic acid sequence which may comprise SEQ ID No. 1, afunctional variant, part or homolog thereof operably linked to aregulatory sequence into a plant wherein said plant is not rice. In oneembodiment, the transgenic plant expresses a nucleic acid sequence whichmay comprise or consist of SEQ ID No. 1.

The plant is characterised in that it shows increased yield, growth,nitrogen transport, nitrogen acquisition, nitrogen stress tolerance,pathogen resistance and/or nitrogen use efficiency.

The term “functional variant of a nucleic acid sequence” as used hereinwith reference to SEQ ID No. 1 or another sequence refers to a variantgene sequence or part of the gene sequence which retains the biologicalfunction of the full non-variant sequence, for example confers increasedgrowth or yield when expressed in a transgenic plant. A functionalvariant also may comprise a variant of the gene of interest which hassequence alterations that do not affect function, for example innon-conserved residues. Also encompassed is a variant that issubstantially identical, i.e. has only some sequence variations, forexample in non-conserved residues, compared to the wild type sequencesas shown herein and is biologically active.

Thus, specifically included in the scope is a functional part of anucleic acid sequence as used herein with reference to SEQ ID No. 1 oranother sequence which retains the biological function of the fullnon-variant sequence, for example confers increased growth or yield whenexpressed in a transgenic plant.

Thus, it is understood, as those skilled in the art will appreciate,that the aspects of the invention, including the methods and uses,encompasses not only a nucleic acid sequence which may comprise orconsisting or SEQ ID No. 1, but also functional variants or parts of SEQID No. 1 that do not affect the biological activity and function of theresulting protein. Alterations in a nucleic acid sequence which resultin the production of a different amino acid at a given site that dohowever not affect the functional properties of the encoded polypeptide,are well known in the art. For example, a codon for the amino acidalanine, a hydrophobic amino acid, may be substituted by a codonencoding another less hydrophobic residue, such as glycine, or a morehydrophobic residue, such as valine, leucine, or isoleucine. Similarly,changes which result in substitution of one negatively charged residuefor another, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a functionally equivalent product. Nucleotidechanges which result in alteration of the N-terminal and C-terminalportions of the polypeptide molecule would also not be expected to alterthe activity of the polypeptide. Each of the proposed modifications iswell within the routine skill in the art, as is determination ofretention of biological activity of the encoded products.

A functional variant of SEQ ID No. 1 has at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the aminoacid represented by SEQ ID NO: 1. A functional variant of SEQ ID NO. 3has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overallsequence identity to the amino acid represented by SEQ ID No: 3. Afunctional variant retains the pH sensing motif.

A functional homolog of SEQ ID No. 1 is a nucleic acid encoding aNRT2.3b peptide which is biologically active in the same way as SEQ IDNo. 1, in other words, for example it confers increased yield or growth.The term functional homolog includes OsNRT2.3b orthologs in other plantspecies.

The homolog of a OsNRT2.3b polypeptide has, in increasing order ofpreference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identityto the amino acid represented by SEQ ID No: 3. Preferably, overallsequence identity is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%. In another embodiment, the OsNRT2.3b nucleic acid sequence has, inincreasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%overall sequence identity to the nucleic acid represented by SEQ IDNo: 1. Preferably, overall sequence identity is 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%. The overall sequence identity is determinedusing a global alignment algorithm known in the art, such as theNeedleman Wunsch algorithm in the program GAP (GCG Wisconsin Package,Accelrys).

Preferably, the OsNRT2.3b homolog/ortholog has the pH sensing motifVYEAIHKI on the cytoplasmic side. In one embodiment, the homolog of aOsNRT2.3b polypeptide has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% overall sequence identity to the amino acid represented by SEQ IDNo: 3 and may comprise the pH sensing motif VYEAIHKI (SEQ ID No. 16).Functional variants or parts of the homologs, for examples as shown inSEQ ID No. 6-15, are also included in the scope of the invention.

FIG. 24 shows examples of homologs/orthologs which have the pH sensingmotif identified in OsNRT2.3b. Thus, preferred orthologous genes orpeptides used according to the various aspects of the invention areselected from the orthologous listed in FIG. 24, including barley,maize, soybean, Brachypodium (SEQ ID Nos. 6-15) and wheat. Variants ofthese sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% to the sequences listed in SEQ ID NO. 6-15 are alsowithin the scope of the invention.

Suitable homologs or orthologs can be identified by sequence comparisonsand identifications of conserved domains. The function of the homolog orortholog can be identified as described herein and a skilled personwould thus be able to confirm the function when expressed in a plant.

For example, according to the various aspects of the invention, anucleic acid encoding an endogenous NRT2.3 peptide may be expressed inany plant as defined herein unless otherwise specified by recombinantmethods. As described above, in certain aspects of the invention, inparticular when the nucleic acid construct may comprise or consists ofSEQ ID No. 1, the plant is not rice. For example, rice OsNRT2.3b may beexpressed in rice and a wheat NRT2.3b may be expressed in wheat.

In another embodiment, a nucleic acid encoding a plant NRT2.3b that isendogenous to a first plant species may be expressed in a second plantusing recombinant methods. For example, a OsNRT2.3b homolog from anotherplant may be expressed in rice.

In one preferred embodiment of the various aspects of the invention,OsNRT2.3b which may comprise SEQ ID No. 1 or a functional variantthereof is expressed in another plant that is not rice. As the inventorshave surprisingly shown, expression of OsNRT2.3b does lead to beneficialphenotypes in other plants that use a different N source. For example,expression may be in a monocot or dicot plant as described herein. Inone embodiment, the plant is wheat or tobacco.

Thus, the invention specifically relates to a method for increasing oneor more of growth, yield, nitrogen transport, NUE, nitrogen acquisition,decreasing photorespiration, increasing intercellular CO₂ levels,increasing photosynthetic efficiency, pathogen resistance andmaintaining/improving pH homeostasis which may comprise introducing andexpressing a nucleic acid sequence which may comprise SEQ ID No. 1, or afunctional variant thereof in another plant that is not rice. Transgenicnon-rice plants expressing a nucleic acid sequence which may compriseSEQ ID No. 1, or a functional variant, part thereof are also encompassedin the scope of the invention, for example wheat or tobacco.

Plants and their endogenous NRT2.3b may be selected from any plant, suchas from one of the families or species listed herein.

Arabidopsis does not have a close relative to OsNRT2.3, the closest isAtNRT2.5, but this does not have a similar pH-sensing motif. A keyaspect of the improved NUE associated with OsNRT2.3b is pH sensitivityof the nitrate transport function. The cytoplasmic pH sensing motif inOsNRT2.3b, that is absent from OsNRT2.3a, provides a link betweennitrogen nutrition and pH regulation. The presence of a pH sensing motifis therefore important for homologs/orthologs in other species.

Homologs/orthologs of OsNRT2.3b can therefore be identified by thepresence of a cytoplasmic pH sensing motif. In one aspect, the inventionrelates to a method for identifying OsNRT2.3b homologs/orthologs inother species which may comprise identifying peptides which may comprisethe cytoplasmic pH sensing motif.

As explained in the examples, when over-expressing OsNRT2.3b in rice,xylem pH was 7 and 7.3 in WT treated respectively with nitrate andammonium, while it was 7.5 and 7.6-7.8 in the OsNRT2.3b over-expressinglines, significantly higher than WT. After 24 h N treatments, phloem sapwas collected. The phloem sap pH was measured and less acidification wasfound in OsNRT2.3b over-expression lines. The difference between WT andover-expression lines was about 0.2 pH units in nitrate and about 0.1 pHunits in ammonium. WT phloem pH decreased from 7.8 to 6.1 and b-S6 from6.7 to 6.0 in nitrate supply from 24 to 48 h treatments; while inammonium treatment WT phloem pH decreased from 7.4 to 6.3 and b-S6 from6.6 to 5.9 from 24 to 48 h. The difference between WT and b-S6 undernitrate supply was remarkably high at 24 h, however no significantdifference was found by 48 h. In ammonium supply, although the pH in WTsap was higher than in b-S6 the difference was not significant. Theacidification of WT phloem pH in nitrate was about 1.7 pH units howeverit was only 0.7 of a pH unit in the b-S6 plants. By 48 h the collectedphloem pH sap had adjusted to give more similar values for WT and b-S6plants (FIG. 17b ). Furthermore the root apoplastic pH in WT and b-S6roots was tested with bromocresol purple indicator¹⁷ after 72 h ofdiffering N treatments. Overexpressing line b-S6 showed alkalinizationin nitrate and acidification in ammonium relative to WT, while the pH inhydroponic medium did not show a significant difference between WT andb-S6 over the same time scale (FIG. 18c ) as the bulk solution was largeenough to buffer any pH changes occurring at the root surface.

The N supply form for plants is well known for influencing plant pHbalance²⁴. The assimilation of ammonium produces at least one H⁺ per NH₄⁺; while NO₃ ⁻ assimilation produces almost one OH⁻ per NO₃ ⁻⁴. EitherH⁺ or OH⁻ produced in excess of that required to maintain cytoplasmic pHare exported from the cell in an energy requiring step (e.g. plasmamembrane H⁺ pumping ATPase)^(4,10). Applicants compared the pH of phloemsap from N-starved rice plants resupplied with nitrate or ammonium.Nitrate and ammonium supply acidified the phloem pH of WT and transgenicplants (FIG. 3d, e ). Interestingly, the phloem acidification wassignificantly lower in the four transgenic lines when compared with WT(FIG. 3d, e ) although no significant difference in nitrateconcentration could be detected in phloem (data not shown). These datashow that transgenic plants are better able to regulate phloem pH.Furthermore the phloem pH difference between WT and transgenics (FIG.17a, b ) could explain the enhanced P and Fe accumulation in leaves ofthe OsNRT2.3b over-expressing plants (FIG. 19). The more acidic phloemsap (FIG. 17) will benefit P and Fe translocation to the leaf²⁵.Together with enhanced N acquisition this was also an important factorfor the plant growth and yield increase.

It has been reported that cytosolic pH acidification inactivatedtransport of aquaporin in oocytes²⁶. Furthermore as nitrate assimilationdepends on photorespiration²⁷, the relationship^(4,28) between theassimilation of nitrate, ammonium and photorespiration is closelycoupled to the shuttling of malate between the cytoplasm and chloroplastto balance pH²⁹.

In plants, the regulation of pH is a requirement that arises for avariety of reasons. The most basic reason is that water spontaneouslyionizes with the consequence that protons cannot be removed entirelyfrom a given solution. Unlike other ions, protons can be consumed or areproduced in certain chemical reactions, with the result that the kind ofnutrition determines to what extent protons may become a problem, oreven a hazard, to the organism. The exact regulatory determinants andcausalities are difficult to analyse (at a given moment) for anysituation because pH influences a great variety of processes in a planttissues and cells and intracellular compartments, and at the same timeH+ activity may be changed by the same processes. The ability to reversea pH perturbation, as well as the extent and the velocity at which thisis accomplished, defines the quality of pH regulation.

The homeostatic maintenance of cytoplasmic pH is important forenergizing the cellular uptake and storage of nutrients and secondarymetabolites because proton-coupled transport systems mediate thesecellular processes. The pH gradients between cellular compartments andthe external environment provide an energy source for these importantprocesses. Many key cellular processes are therefore enhanced by theimproved pH homeostasis associated with a mixed nitrate and ammoniumnitrogen supply.

Applicants have shown that the OsNRT2.3b may comprise a pH sensing motifon the cytosolic side of the plasma membrane which is not present inOsNRT2.3a on the cytosolic side. The pH-sensing motif VYEAIHKI (SEQ IDNo. 16) around histidine residue 167 of OsNRT2.3b which faces thecytosolic side of the plasma membrane is a characteristic of the anionexchanger family, which is found in many different organisms includingmammals and may therefore be of more general biological significance. Asdemonstrated in the examples, Applicants have shown that after a singleamino acid mutation (H167R), OsNRT2.3b lost this function of cytosolicpH regulation, even after repeated cycles of nitrate treatment (FIG. 5b).

The OsNRT2.3b sensing motif regulates the cytosolic pH in the plant.

Applicants have also shown that the pH sensing motif of OsNRT2.3b isimportant for these effects in rice by linking the plant's pH status tonitrate supply.

In yet another aspect, the invention therefore relates to a method forregulating pH homeostasis which may comprise introducing and expressinga nucleic acid construct which may comprise a nucleic acid sequencewhich may comprise SEQ ID No. 1 operably linked to a regulatory sequencein a plant. In one aspect, the plant is not rice.

In a further aspect, the invention relates to a method for reducingacidification in a plant which may comprise introducing and expressing anucleic acid construct which may comprise a nucleic acid sequence whichmay comprise SEQ ID No. 1 operably linked to a regulatory sequence in aplant. In one aspect, the plant is not rice.

Acidification may be reduced by at least about 0.1 pH units, for example0.1, 0.2. 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or more.

In a further aspect, the invention relates to a method for alteringnitrate transport and pH homeostasis in a plant which may compriseintroducing and expressing a nucleic acid construct which may comprise anucleic acid sequence which may comprise SEQ ID No. 1 operably linked toa regulatory sequence in a plant wherein said nucleic acid may comprisea mutation in the pH sensing motif VYEAIHKI (SEQ ID No. 16). Themutation renders the pH sensing motif non-functional.

As set out elsewhere herein, the regulatory sequence may be aconstitutive promoter as described herein or a tissue specific promoter.In one embodiment, the promoter is a phloem specific promoter asdescribed herein.

The term plant is also defined elsewhere herein. Preferably, the plantis a crop plant. Most preferred plants are maize, rice, wheat, oilseedrape, sorghum, soybean, potato, tomato, grape, barley, pea, bean, fieldbean, lettuce, cotton, sugar cane, sugar beet, tobacco, broccoli orother vegetable brassicas or poplar. In one embodiment, the plant is notrice.

The invention also relates to the use of a nucleic acid which maycomprise SEQ ID No. 1, a functional variant, part or homolog thereofencoding SEQ ID No 3, a functional variant, part or homolog thereofwhich may comprise the pH sensing motif VYEAIHKI (SEQ ID No. 16) inregulating pH in a transgenic plant.

In another aspect, the invention relates to a method for increasinggrowth of a plant which may comprise introducing and expressing anucleic acid construct which may comprise a nucleic acid sequence asdefined in SEQ ID No. 1 operably linked to a regulatory sequence into aplant wherein said regulatory sequence is a constitutive promoter or aphloem specific promoter and wherein said plant does not overexpress anucleic acid sequence which may comprise SEQ ID No. 2.

In another aspect, the invention relates to a method for increasingnitrogen use efficiency of a plant which may comprise introducing andexpressing a nucleic acid construct which may comprise a nucleic acidsequence which may comprise SEQ ID No. 1 operably linked to a regulatorysequence into a plant wherein said regulatory sequence is a constitutivepromoter or a phloem specific promoter and wherein said plant does notoverexpress a nucleic acid sequence which may comprise SEQ ID No. 2.

In another aspect, the invention relates to a method for improving yieldof a plant which may comprise introducing and expressing a nucleic acidconstruct which may comprise a nucleic acid sequence which may compriseSEQ ID No. 1 operably linked to a regulatory sequence into a plantwherein said regulatory sequence is a constitutive promoter or a phloemspecific promoter and wherein said plant does not overexpress a nucleicacid sequence which may comprise SEQ ID No. 2.

In another aspect, the invention relates to a method for increasingnitrate transport in a plant which may comprise introducing andexpressing a nucleic acid construct which may comprise a nucleic acidsequence which may comprise SEQ ID No. 1 operably linked to a regulatorysequence into a plant wherein said regulatory sequence is a constitutivepromoter or a phloem specific promoter and wherein said plant does notoverexpress a nucleic acid sequence which may comprise SEQ ID No. 2.

In another aspect, the invention relates to a method for increasingnitrogen acquisition of a plant which may comprise introducing andexpressing a nucleic acid construct which may comprise a nucleic acidsequence which may comprise SEQ ID No. 1 operably linked to a regulatorysequence into a plant wherein said regulatory sequence is a constitutivepromoter or a phloem specific promoter and wherein said plant does notoverexpress a nucleic acid sequence which may comprise SEQ ID No. 2.

In one embodiment of the various methods described herein for increasingNUE, growth, yield, nitrogen acquisition and/or nitrate transport, saidtraits are increased under stress conditions, for example nitrogenstress.

Thus, in another aspect, the invention relates to a method forconferring tolerance to nitrogen stress to a plant which may compriseintroducing and expressing nucleic acid construct which may comprise anucleic acid sequence which may comprise SEQ ID No. 1 operably linked toa regulatory sequence into a plant wherein said regulatory sequence is aconstitutive promoter or a phloem specific promoter and wherein saidplant does not overexpress a nucleic acid sequence which may compriseSEQ ID No. 2.

Thus, in another aspect, the invention relates to a method forconferring pathogen resistance to a plant which may comprise introducingand expressing nucleic acid construct which may comprise a nucleic acidsequence which may comprise SEQ ID No. 1 operably linked to a regulatorysequence into a plant wherein said regulatory sequence is a constitutivepromoter or a phloem specific promoter and wherein said plant does notoverexpress a nucleic acid sequence which may comprise SEQ ID No. 2. Ifthe plant is rice, then the pathogen may be Fusarium wilt, Leaf blightand Stripe rust.

According to the methods above, the regulatory sequence according to themethod and plants above is as described herein and may therefore be aconstitutive promoter as described herein, an inducible promoter or atissue specific promoter. In one embodiment, the promoter is a phloemspecific promoter as described herein. Phloem-specific expression may beimportant for the function of the OsNRT2.3b, as the vascular tissue isimportant for pH regulation and it has recently been shown that nitratetransport in the phloem occurs in plants and may be a significant routefor nitrogen delivery to the shoot.

The term plant is also defined elsewhere herein. Preferably, the plantis a crop plant. Most preferred plants are maize, rice, wheat, oilseedrape, sorghum, soybean, potato, tomato, grape, barley, pea, bean, fieldbean, lettuce, cotton, sugar cane, sugar beet, broccoli or othervegetable brassicas or poplar. In one embodiment, the plant is not rice.

In another aspect, the invention relates to a method for increasingnitrogen use, yield, NUE, nitrogen efficiency, tolerance to nitrogenstress, pathogen resistance, nitrogen acquisition and/or nitratetransport of a plant which may comprise introducing and expressingnucleic acid construct which may comprise a nucleic acid sequence whichmay comprise SEQ ID No. 1 operably linked to a phloem specific promoterin a plant. The term plant is also defined elsewhere herein. Preferably,the plant is a crop plant. Most preferred plants are maize, rice, wheat,oilseed rape, sorghum, soybean, potato, tomato, grape, barley, pea,bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli orother vegetable brassicas or poplar. In one embodiment, the plant is notrice.

In another aspect, the invention relates to a method for making atransgenic plant having increased yield, growth and/or nitrogen useefficiency which may comprise introducing and expressing in a plant orplant cell a nucleic acid construct which may comprise a nucleic acidsequence as defined in SEQ ID No. 1 operably linked to a regulatorysequence wherein said regulatory sequence is a constitutive promoter ora phloem specific promoter and wherein said plant does not overexpress anucleic acid sequence which may comprise SEQ ID No. 2.

The method further may comprise regenerating a transgenic plant from theplant or plant cell after step a) wherein the transgenic plant maycomprise in its genome SEQ ID No. 1 operably linked to a regulatorysequence and obtaining a progeny plant derived from the transgenic plantwherein said progeny plant exhibits increased yield, growth and/ornitrogen use efficiency. These methods are carried out as describedelsewhere herein.

Plants or parts thereof obtained or obtainable by the method for makinga transgenic plant as described above are also within the scope of theinvention.

In another aspect, the invention relates to a transgenic plantexpressing a nucleic acid construct which may comprise a nucleic acidsequence as defined in SEQ ID No. 1 operably linked to a regulatorysequence into a plant wherein said regulatory sequence is a constitutivepromoter or a phloem specific promoter and wherein said plant does notoverexpress a nucleic acid sequence SEQ ID No. 2.

Plants that can be used according to these methods of the invention arespecifically listed elsewhere herein but also include rice. Preferably,the plant is a crop plant or biofuel plant as defined elsewhere herein.

Most preferred plants are rice, maize, wheat, oilseed rape, sorghum,soybean, potato, tomato, tobacco, grape, barley, pea, bean, field bean,lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetablebrassicas or poplar.

In one embodiment, the plant is wheat and the promoter is a phloemspecific promoter as described herein. In one embodiment, the plant istobacco and the promoter is a phloem specific promoter as describedherein.

The plant is characterised in that it shows having increased yield,growth, nitrogen transport, nitrogen acquisition, nitrogen stresstolerance and/or nitrogen use efficiency.

Other objects and advantages of this invention will be appreciated froma review of the complete disclosure provided herein and the appendedclaims.

While the foregoing disclosure provides a general description of thesubject matter encompassed within the scope of the present invention,including methods, as well as the best mode thereof, of making and usingthis invention, the following examples are provided to further enablethose skilled in the art to practice this invention and to provide acomplete written description thereof. However, those skilled in the artwill appreciate that the specifics of these examples should not be readas limiting on the invention, the scope of which should be apprehendedfrom the claims and equivalents thereof appended to this disclosure.Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.The specifics of these examples should not be treated as limiting.

All documents mentioned in this specification, including references todatabases for gene or protein sequences, are incorporated herein byreference in their entirety.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES

The invention is further described in the following non-limitingexamples

1. Expression of OsNRT2.3a and OsNRT2.3b in Rice

Materials and Methods

Over-expression Vector Construction and Transgenic Plants

The open reading frames of OsNRT2.3a and OsNRT2.3b were amplified bygene specific primers. The fragment was treated with restriction enzymesand inserted in vectors and sequenced before transformation. Rice (Oryzasativa) embryonic calli were transformed using Agrobacterium-mediatedmethods³³. One copy insertion T0 plants were harvested and grown togenerate T1 plants. Homozygous T1 plants were taken for T2 generation.Two lines of T2 OsNRT2.3a over-expression plants, a-U1 and a-U2 and fourlines of T2 OsNRT2.3b over-expression plants, b-U1, b-U2, b-S2 and b-S6were used for further experiments. T2 field experiments were conductedin Changxing experiment station of Zhejiang University (May-October2010) in four N application N as urea levels as 0, 75, 150 and 300 kgN/ha. Seeds were germinated on 5th May and seedlings of each type wereplanted at 3 rows and 33 plants with 25 cm (row space)×20 cm (plantspace) on 5th June. Plants were grown in blocks (FIG. 23a,b ) with arandom order for each N application. For the large scale experiments at75 kg N/ha, the plants were transplanted as 10 rows×128 plants (FIG. 23d). Three replications were used for all field experiments and the plotswere finally harvested on the 10th October. The soil nutrient status inthis experiment station was total nitrogen (N): 1.00±0.18 mg/g, totalphosphorus (P) 0.38±0.08 mg/g, total potassium (K) 39±2.3 mg/g, Olsen P(0.5 mM NaHCO₃-extractable P) 23±4.1 mg/kg and soil pH was 6.3±0.47(n=6). 60 kg P (as Ca(H₂PO₄)₂)/ha and 110 kg K (as K₂SO₄)/ha fertilizerwas applied to the paddy before transferring the rice seedlings. Thefirst N application was carried out before transferring on 3th June and20% total N fertilizer was mixed into soil. Second application was 40%on 12 June when the rice was at the beginning of tilling stage. Thefinal application was 40% on 20 June. The rice growth period atChangxing was 120 ±3 days for WT, a-U1 and a-U2 lines, and 130±2 days at0-75 kg N/ha level, 135±2 days at 150 kg N/ha level and 140±2 days at300 kg N/ha level for b-U1, b-U2, b-S2 and b-S6 lines. The grain yieldwas measured at harvest and NUE was defined as grain yield perfertilizer N applied. For the ¹⁵N uptake, xylem and phloem sapcollection experiments, hydroponic growth conditions were used asdescribed previously³⁴ in IRRI culture medium at pH 5.5 with 1.25 mMNH₄NO₃ as the N supply unless stated otherwise. Roots RNA was abstractedfor RT-PCR analysis.

Antibody Production and Western Blot

The full cDNA sequences of OsNRT2.3a/b genes were amplified fromplasmids of OsNRT2.3a (AK109776) and OsNRT2.3b (AK072215) by primers, F:GGAATTCTCACACCCCGGCCGG (SEQ ID No. 17), R: CGGGATCCATGTGGGGC GGCATGCTC(SEQ ID No. 18). The plasmids were kindly provided by Dr. Kikuchi(KOME). The PCR fragment was sub-cloned into the bacterial expressionvector pGSX (Amersham) at BamH I and EcoR I sites. The amino acidproducts were purified and their monoclonal-antibodies weresynthesized³⁵. The monoclonal-antibody was selected from 192 individualcell specific reactions to OsNRT2.3a (516 aa) or OsNRT2.3b (486 aa)protein. Plasma membrane protein abstraction from roots and western blotwas done as previously described^(10,14) and repeated twice.

RNA In Situ Hybridization

RNA in situ hybridization was performed as previously described³⁶. ForOsNRT2.3b probe, the binding site is in OsNRT2.3b specific 5′ UTR withits sequence CGATGGTTGGGTGCGGCGAGA (SEQ ID No. 19). The nonsensesequence is GCTACCAACCCACGCCGCTCT (SEQ ID No. 20). All probes werelabeled at 5′ end with DIG.

Determination of Root ¹⁵N Accumulation

Rice seedlings of WT and over-expression plants were grown in IRRInutrient solution containing 1.25 mM NH₄NO₃ for two months in greenhouseand then deprived of N for 3 days. The plants were rinsed in 0.1 mMCaSO₄ for 1 min, then transferred to the solution containing either 1.25mM Ca(¹⁵NO₃)₂ (atom % ¹⁵N: 99.27%) or (¹⁵NH₄)₂SO₄ (atom % ¹⁵N: 95.7%) or¹⁵NH₄NO₃ (atom % ¹⁵N: 45%) or NH₄ ¹⁵NO₃ (atom % ¹⁵N: 45.25%) or ¹⁵NH₄¹⁵NO₃ (atom % ¹⁵N: 95.5%) for 5 min and finally rinsed again in 0.1 mMCaSO₄ for 1 min. Roots were separated from the shoots immediately afterthe final transfer to CaSO₄, and frozen in liquid N. After grounding, analiquot of the powder was dried to a constant weight at 70° C. 10 mgpowder of each sample was analyzed using the MAT253-Flash EA1112-MSsystem (Thermo Fisher Scientific, Inc., USA). The whole experiment wasrepeated twice and each time with five replicates.

Xylem and Phloem Sap Collection

Rice seedlings were grown in 1.25 mM NH₄NO₃ for 8 weeks and thentransferred to N treatments (nitrate: 1.25 mM Ca(NO₃)₂; ammonium: 1.25mM (NH₄)₂SO₄) for 24 h and then cut at 4 cm above root. The below in Nsolutions was for xylem sap collection³⁴ and the top was for phloem sapcollection¹⁶.

For phloem sap collection, briefly each shoot was put into a 50 ml glasstubes with 15 ml 25 mM EDTA-Na₂ covered with Parafilm. The shoot wasinserted through the Parafilm and phloem sap was collected for 24 h.Phloem pH changes were measured using a pH meter (model 868, ThermoOrion, USA) and by calculation of the pH difference in samples at thestart and end of the phloem sap collection period. The experiment wasconducted with 5 replicate samples and was repeated twice.

Phloem sap was also collected using an insect feeding method with thesame plants as above. Each plant was set in a 250 ml bottle of IRRInutrient solution with six plants kept in the insect cage at 26 C and a16 h light period. Seven to ten adult brown plant hopper adults weretransferred on to each plant at the beginning of the N treatments. Ricephloem honey dew secreted by the insects was collected at 24 h, 48 hduration of N treatments (FIG. 17).

Oocyte Preparation, mRNA Injection, ¹⁵N Uptake and Electrophysiology

Oocytes preparation, mRNA injection, ¹⁵N-nitrate uptake andelectrophysiology have been described previously³⁷⁻³⁹. 0.5 mM Na¹⁵N—NO₃or ¹⁵N—NH₄Cl in ND96 was used for ¹⁵N uptake experiment for 16 h³⁸. ThepH selective microelectrode method was used to measure cytosolic pH¹⁸.

Single Site Mutation of OsNRT2.3b and mRNA Synthesis

A point mutation (H167R) of OsNRT2.3b was generated using a PCR method.The point mutant was processed by PCR two fragments of OsNRT2.3b withthe mutant site and new restriction site in the primers. OsNRT2.3b cDNAin pT7Ts was used as a DNA template and the first PCR fragment (H167RB)was sub-cloned into HindIII and XbaI of pT7Ts. New plasmid and secondPCR fragment (H167R) were digested by Csp45 I and Xba I and ligated intothe final plasmid with H167R site mutated OsNRT2.3b cDNA (pH167R). ThemRNA synthesis of pH167R was described as above.

RNA Preparation and DNA Microarray Hybridization

Three replicates each of WT (Nipponbare), a-U1 and b-S6 shoots wereharvested from 150 Kg N/ha treatment in field of Changxing experimentstation at 10:00 am of the 1th August i.e. the maximum tillering stagefor all plants. Shoot tissues samples taken for RNA extraction wereflash frozen at −80° C. in liquid nitrogen immediately on harvesting.RNA extraction, hybridization with Affymetrix rice GeneChip arrays(Santa Clara, Calif., USA), data analyses and annotation were asdescribed in previous reports⁴⁰.

Quantitative Real-time RT-PCR

Total RNA from three biological representatives, specifically from theroots and shoots of WT and transgenic plants, was isolated using theTRIzol reagent according to the manufacturer's instructions (InvitrogenLife Technologies, Carlsbad, Calif., USA)¹³.

Gas Exchange and Postillumination CO₂ Burst Measurements

The rate of light-saturated photosynthesis of flag leaves was measuredfrom 9:00 h to 15:00 h using a Li-Cor 6400 portable photosynthesis opensystem at the plants in 150 Kg N/ha treatment in field of Changxingexperiment on the same day as microarray sampling. Leaf temperatureduring measurements was maintained at 27.0±0.1° C. with a photosyntheticphoton flux intensity (PPFD) of 1500 μmol photons m⁻²·s⁻¹ as describedbefore⁴¹. The ambient CO₂ concentration in the cuvette (Ca-c) wasadjusted as atmospheric CO₂ concentration (Ca) (417±1.0 μmol CO₂ mol⁻¹),and the relative humidity was maintained at 20%. Data were recordedafter equilibration to a steady state (10 min). The measured leaves werelabelled, and leaf areas were calculated based on the labelled area. Thepostillumination CO₂ burst (PIB) was measured at the same labelled leafunder photorespiratory conditions (saturating PPFD of 1,500 μmol photonsm⁻²·s⁻¹, Ca-c CO₂ concentration of 100 μmol CO₂ mol⁻¹, relative humidityof 60%-70%) as described before²².

Results

Over-expression of OsNRT2.3b Increased Rice Growth

Applicants generated rice (Oryza sativa L ssp. Japonica, cv. Nipponbare)plants that over-express OsNRT2.3a and OsNRT2.3b byAgrobacterium-mediated transformation, using either ubiquitin or 35Spromoters (FIG. 1a, b ). The over-expression lines were named a-U1 anda-U2 for OsNRT2.3a, b-U1, b-U2, b-S2 and b-S6 for OsNRT2.3b,respectively with one copy insertion. Interestingly, the OsNRT2.3bover-expression lines, which were confirmed at both transcript andprotein levels (FIG. 1 cd), showed more growth compared with wild type(WT) (FIG. 1a, b ). The biomass and panicle size of over-expressionlines was greater than WT (FIG. 11; Table 2-3). The primary and secondrachis size was increased therefore the total number of seeds perpanicle was greater than WT (FIG. 11, Table 2). By contrast, theOsNRT2.3a over-expression plants did not show visible difference from WTeven though OsNRT2.3a mRNA and protein was increased in the transformedlines (FIG. 1c, d , FIG. 11). The in situ hybridization results showedthat OsNRT2.3b mRNA in b-S6 leaf was over-expressed in the epidermal,phloem and mesophyll cells when compared with wild type (FIG. 1e ).Furthermore, when OsNRT2.3b was over-expressed in other high yieldingand high NUE rice cultivars, WYJ7 from southern China and YF47 fromnorthern China, their grain yield and NUE (grain yield divided by the Nfertilizer applied) were also significantly increased (FIGS. 12, 13).

Field Trials of Over-expression Lines Show Increased Grain Yield and NUEin both Subtropical and Tropical Climates at a Range of N FertilizationRates

Encouraged by the strong phenotypes of the OsNRT2.3b over-expressingplants in hydroponics and soil pots, Applicants grew selectedNipponbare, WYJ7 and YF47 transgenic lines and their wild types in 4field trials to evaluate their performance under different fertilizer Nrates.

Four Nipponbare T2 transgenic lines and WT were grown with four levelsof N fertilizer application in a paddy field (soil pH 6.3) located atChangxing in the subtropical climate region (FIG. 2). Compared with WT,biomass, seed numbers per panicle, ripening rate, grain yield and NUE ofthe transgenic lines were significantly increased at all levels of Napplication (FIG. 2a-c , Table 5). The average increase in grain yieldranged from 33% at 75 kg N/ha to 25% at 300 kg N/ha. The grain yield ofthe over-expression lines supplied with 150 kg N/ha was 6% to 13% higherthan that of WT yield fertilized with 300 kg N/ha (FIG. 2b ). Remarkablythe best performing transgenic line, b-S6 produced similar grain yieldat 75 kg N/ha to WT at 300 kg N/ha (FIG. 2b , Table 5). The NUE of theOsNRT2.3b over-expressing lines reached 68-79 g/g N at the 75 kg N/haapplication level, compared with 55 g/g N in WT (FIG. 2c ). In a largescale field experiment supplied with 75 kg N/ha, the yield and NUE ofthe line b-U2 were 30.5% more than WT; while for line b-S6 the valueswere even greater at 40.5% (FIGS. 2 d,e,f).

In a second field trial, the T5 generations of b-S2 and b-S6 were grownin tropical Hainan (FIG. 14a ). Significant increases in grain yield andNUE were again obtained. The largest difference between the transgeniclines and Nipponbare WT was found in the 110 kg N/ha supply (FIG. 14b ).Furthermore, crossing b-S6 T5 plants with WT confirmed that the b-S6phenotype was completely contributed by OsNRT2.3b over-expression as inF2 generation plants the aa genotype returned to WT and AA genotype waslike b-S6 (FIG. 15).

The third field trial tested the OsNRT2.3b over-expressing lines in theWYJ7 background with three N supplies (110 and 220 kg N/ha) inChangxing. Among the four transformed lines (T2 generation), grain yieldwas 35-51% larger than WT at 110 kg N/ha and 38-42% larger at 220 kgN/ha. On average, the NUE was 43% higher than WT (FIG. 12f ).

The fourth field trial tested the OsNRT2.3b over-expressing lines in theYF47 cultivar background in Hainan (FIG. 15). Similar to the resultsobtained with the other two backgrounds, OsNRT2.3b over-expression inYF47 generated more biomass and 39% more grain yield than WT at a usualN fertilizer supply (150 kg/ha) (FIG. 13a, d ). Taken together,OsNRT2.3b over-expression produced consistent effects on grain yield andNUE across different cultivar backgrounds, climates, and N applicationrates.

In the Nipponbare background, OsNRT2.3b over-expression resulted in adelay in flowering compared with WT, by 15±2 days at 150 kg/ha and 20±2days at 300 kg/ha (FIGS. 2a,d , FIG. 16, Table 1). In pot experimentswith these plants, at 120 days after germination the grain yield of b-S2and b-S6 was 37% and 40% higher than WT (FIG. 16g ). At 140 days thegrain yield of b-S2 and b-S6 was 55% and 49% higher than WT (FIG. 16g ).The extra 20 days increased their yield by only 18% and 9% compared withthe data at the first 120 days. It was clear that the greatestcontribution of OsNRT2.3b over-expression to grain yield occurred at 120days. Furthermore the 20 days growth delay did not significantlyincrease the total N uptake for OsNRT2.3b over-expression plants (FIG.16h ). However 20 days delay increased the ratio of biomass and Ntransfer to the grain (Tables 3-4) and the N utilization (assimilation)efficiency (NUtE) from 33 g-grains/g-N at 120 days (no significantdifference from WT) to 39.1-40.2 g-grains/g-N at 140 days (a significantincrease relative to WT for b-S2 and b-S6 (FIG. 16i ). In fact relativeto WT there was no flowering delay of OsNRT2.3b over-expressers in WYJ7and YF47 background.

OsNRT2.3b Over-expression Increased Nitrate Influx, Transport to Shoot,Xylem pH, Phloem pH Homeostasis, P and Fe Accumulation in Leaves.

Applicants measured the effect of OsNRT2.3b over-expression on¹⁵N-nitrate influx in four Nipponbare transformed lines hydroponicallygrown at pH 6 (FIG. 3a ). The nitrate influx rate was increasedsignificantly in all the transgenic lines compared with WT (FIG. 3a ),demonstrating increased activities of OsNRT2.3b in these plants. Bycontrast, OsNRT2.3b over-expression had no significant effect on theshort-term ¹⁵N-ammonium uptake (FIG. 3a ).

More nitrate and less ammonium were detected in the xylem of b-U1, b-U2,b-S2 and b-S6 in compared with WT under nitrate supply (FIG. 3b ). XylempH was 7 and 7.3 in WT treated respectively with nitrate and ammonium,while it was 7.5 and 7.6-7.8 in the OsNRT2.3b over-expressing lines,significantly higher than WT. After 24 h N treatments, phloem sap wascollected. The phloem sap pH was measured using the EDTA-Na₂ collectionmethod¹⁶ and less acidification was found in OsNRT2.3b over-expressionlines. The difference between WT and over-expression lines was about 0.2pH units in nitrate and about 0.1 pH units in ammonium. To check thephloem pH using a different method, the sap was collected fromphloem-feeding insects (described in FIG. 9). WT phloem pH decreasedfrom 7.8 to 6.1 and b-S6 from 6.7 to 6.0 in nitrate supply from 24 to 48h treatments (FIG. 9a ); while in ammonium treatment WT phloem pHdecreased from 7.4 to 6.3 and b-S6 from 6.6 to 5.9 from 24 to 48 h (FIG.9b ). The difference between WT and b-S6 under nitrate supply wasremarkably high at 24 h, however no significant difference was found by48 h. In ammonium supply, although the pH in WT sap was higher than inb-S6 the difference was not significant (FIG. 9b ). The acidification ofWT phloem pH in nitrate was about 1.7 pH units however it was only 0.7of a pH unit in the b-S6 plants (FIG. 3e ). By 48 h the collected phloempH sap had adjusted to give more similar values for WT and b-S6 plants(FIG. 9b ). Furthermore the root apoplastic pH in WT and b-S6 roots wastested with bromocresol purple indicator¹⁷ after 72 h of differing Ntreatments. Overexpressing line b-S6 showed alkalinization in nitrateand acidification in ammonium relative to WT (FIG. 18a, b ), while thepH in hydroponic medium did not show a significant difference between WTand b-S6 over the same time scale (FIG. 18c ) as the bulk solution waslarge enough to buffer any pH changes occurring at the root surface.

Under ammonium nitrate supply the total P and Fe in the plants were alsomeasured. Both total P and Fe were increased in the leaves of theover-expressing lines compared with WT (FIG. 19), especially for totalFe, it was 3-6 times more than WT.

OsNRT2.3b Over-expression Increased Total N Uptake in Mixture Supply ofAmmonium and Nitrate at pH 4 and 6.

N-starved plants were resupplied with NH₄ ¹⁵NO₃ or ¹⁵NH₄NO₃ or ¹⁵NH₄¹⁵NO₃ in pH 4, and 6 for 5 min to measure N uptake by root (FIG. 4).These results clearly showed as the pH increased, the ¹⁵NO₃ ⁻ influx wasdecreased, ¹⁵NH₄ ⁺ and total ¹⁵N was increased dramatically for both WTand all the OsNRT2.3b transgenic lines (comparing FIGS. 4 a, b, c). TheOsNRT2.3b over-expression lines showed more ¹⁵NH₄NO₃ and total N uptakeat pH 4 and 6. In the field experiments, soil pH ranged from 4.4 to 6.4(FIGS. 1, 2, 13-14), the phenotype of the field grown transgenic linescan be explained by the enhanced total N acquisition (nitrate andammonium) of these plants.

Transport Function of OsNRT2.3b Regulated by Cytosolic pH

As over-expression of OsNRT2.3b has such a major impact on NUE andgrowth of rice and this effect was related to plant pH homeostasis,Applicants investigated the transporter function in more detail at themolecular level. In heterologous expression experiments thenitrate-elicited changes in membrane potential of Xenopus oocytesexpressing OsNRT2.3b could not respond to sequential nitrate treatments(FIG. 5a ). It was necessary for an oocyte to rest for at least 30 minbetween nitrate treatments to recover the electrical response or itcould respond to nitrate immediately after washing with pH 8.0 saline(FIG. 5a ). Double-barrelled pH electrode measurements showed that a 0.2pH acidification of cytosolic pH prevented the second nitrate responseof OsNRT2.3b injected oocytes (FIG. 5a ). A slight delay of cytosolic pHresponse was observed compared with membrane potential shift to externalnitrate treatment (FIG. 5a ). This cytosolic pH delay from membranepotential response was presented by other authors^(18,19).

The consensus transmembrane (TM) secondary structure of OsNRT2.3b waspredicted using software packages. 14 software packages predicted thatOsNRT2.3 has 11 TM with the N terminus on the cytosolic side and thefirst 5 TM are presented in table below. H 167 amino acid was predictedin the cytosolic side in both table and figure, which was shown with thesingle site mutagenesis target ringed in prediction secondary structurebelow, predicted by http://bioinfo.si.hirosaki-u.ac.jp/˜ConPred2/.). ThepH-sensing motif VYEAIHKI is around residue 167 on the cytosolic side.

Interestingly, bioinformatics analysis of the predicted OsNRT2.3bprotein structure revealed a pH-sensing motif VYEAIHKI²⁰ around ahistidine (H) residue of OsNRT2.3b which faces the cytosolic side of theplasma membrane After a single site mutation (H167R), OsNRT2.3b lostthis function of cytosolic pH regulation, even after repeated cycles ofnitrate treatment (FIG. 5b ). The results show that endogenous oocytecellular pH homeostatic mechanisms were able to restore cytosolic pHabove the threshold for OsNRT2.3b transport activity. When oocytes wereincubated in ¹⁵N-nitrate for only 4 hours, the regulatory effect ofcytosolic pH on nitrate transport was clear, as the comparison of H167Rand wild type forms of OsNRT2.3b showed that the mutation resulted in amuch larger nitrate accumulation (FIG. 5c ). However, after an 8 hincubation the differences in activity of the two forms of thetransporter had disappeared; suggesting that after the longer incubationthe accumulation of nitrate had reached a maximum in the oocytes.

Decreased Photorespiratory Gene Expression and Photorespiration

Some genes are known to be specifically associated with plantphotorespiratory activity²¹. Microarray and confirmatory qPCRs showed agene expression pattern that indicates that photorespiration was alteredin rice over-expressing OsNRT2.3b, when compared with WT and lines withincreased OsNRT2.3a transcripts.

The total photosynthesis in b-S2 and b-S6 increased compared with WT,but b-U1 and b-U2 did not significantly increase. However intercellularCO₂ concentration was increased and the photorespiratory rate wasdecreased in all over-expression lines compared with WT (FIG. 20). Thereduced photorespiration and enhanced photosynthesis in transgenicplants could contribute more biomass²². These data suggested thatincreased photosynthetic efficiency in plants overexpressing OsNRT2.3bcontributes to the strong phenotype.

Discussion

The pH sensing activity switch of OsNRT2.3b is one of the key factorsproviding an explanation for the phenotype of the transgenic plants,since transforming OsNRT2.3b H167R mutant gene into Nipponbare plantsdid not increase height, yield and did not delay reproductive stage(FIG. 21). The pH-sensing motif VYEAIHKI (SEQ ID No. 16) around residue167 is a characteristic of the anion exchanger family, which is found inmany different organisms including mammals and may therefore be of moregeneral biological significance²⁰. Increasing the external pH decreasednitrate accumulation in the OsNRT2.3b expressing oocytes (FIG. 5d ),supporting the idea that OsNRT2.3b is a proton-nitrate co-transporter¹⁴.Increasing the external pH decreases the proton gradient driving nitratetransport, but on the other hand it restores the nitrate transportfunction of OsNRT2.3b by making the cytosol more alkaline. Both effectsoccur via pH changes, but each happens on different sides of the plasmamembrane. In planta the simultaneous influx of nitrate and ammoniumcounters the cytosolic pH regulatory effect of the OsNRT2.3b sensingmotif. The proton-cotransport mechanism for the entry of nitrate intocells provides a cytosolic acidification, while ammonium transport cancause an alkalinization²³ that may enhances proton-coupled nitratetransport. This short-term synergism between ammonium and nitratetransport to maintain cytosolic pH can explain the measured increase in¹⁵N-ammonium uptake when the plant was supplied with a mixed N source(FIG. 4), with the exclusion of the possibility that OsNRT2.3b proteinitself might uptake ammonium in oocytes (FIG. 22). In WT plants,OsNRT2.3b expression was low¹³ and mainly localized in the phloem ofleaves but not roots (FIG. 1d ). The transgenic plants with OsNRT2.3bover-expression driven by strong promoters had more general tissueexpression (FIG. 1d ). The synergism between ammonium and nitratetransport was enhanced by over-expression of the pH sensing transporterOsNRT2.3b more generally in root cells.

The N supply form for plants is well known for influencing plant pHbalance²⁴. The assimilation of ammonium produces at least one H⁺ per NH₄⁺; while NO₃ ⁻ assimilation produces almost one OH⁻ per NO₃ ⁻⁴. EitherH⁺ or OH⁻ produced in excess of that required to maintain cytoplasmic pHare exported from the cell in an energy requiring step (e.g. plasmamembrane H⁺ pumping ATPase)^(4,10). The vascular specific expression ofOsNRT2.3b in WT plants suggests a possible specific role in longdistance transport within plants. To test this idea Applicants comparedthe pH of phloem sap from N-starved rice plants resupplied with nitrateor ammonium. Nitrate and ammonium supply acidified the phloem pH of WTand transgenic plants (FIG. 3d, e ). Interestingly, the phloemacidification was significantly lower in the four transgenic lines whencompared with WT (FIG. 3d, e ) although no significant difference innitrate concentration could be detected in phloem (data not shown).These data show that transgenic plants are better able to regulatephloem pH, indicating that this is an important factor for the improvedNUE. Furthermore the phloem pH difference between WT and transgenics(FIG. 17a, b ) could explain the enhanced P and Fe accumulation inleaves of the OsNRT2.3b over-expressing plants (FIG. 19). The moreacidic phloem sap (FIG. 17) will benefit P and Fe translocation to theleaf²⁵. Together with enhanced N acquisition this was also an importantfactor for the plant growth and yield increase.

It has been reported that cytosolic pH acidification inactivatedtransport of aquaporin in oocytes²⁶. As discussed by these and otherauthors²⁶, it suggested cytosolic pH could be a key regulation for bothaquaporin and nitrate transporter in plants. Furthermore as nitrateassimilation depends on photorespiration²⁷, the relationship^(4,28)between the assimilation of nitrate, ammonium and photorespiration isclosely coupled to the shuttling of malate between the cytoplasm andchloroplast to balance pH²⁹.

Many important crop traits like NUE are well known to be complexmulti-gene traits⁴. However, a few reports show that changing expressionof a single trans-gene can significantly improve crop NUE³⁰⁻³². Thedramatic enhanced performance of the OsNRT2.3b transformed plants underdifferent field conditions shows the prospects for improving rice NUEthrough single trans-gene approaches. The coupling of pH balance and NUEis likely to have more general relevance to crops and offers a promisingway of improving NUE.

2. Expression of OsNRT2.3b in Arabidopsis

Applicants have obtained data with 35S-driven expression of OsNRT2.3b inArabidopsis. The Arabidopsis plants were transformed using standardfloral-dipping Agrobacterium-mediated transformation techniques (Clough& Bent 1998). In Petri dish growth experiments Arabidopsis plants weresupplied with either 0.2 or 6 mM nitrate supplies. Three independentlines of Arabidopsis plants overexpressing the OsNRT2.3b (checked at themRNA level, using RT-PCR) were tested and compared with wild typecontrol plants (see FIG. 6). The data in FIG. 6 show that threeindependent Arabidopsis lines overexpressing the rice transportergrowing on 6 mM nitrate had significantly more shoot biomass (Fig A) andhad shorter roots on 0.2 mM supply (Fig B) relative to wild type plants.Furthermore, two of these lines accumulated more tissue nitrate.

These plants were grown a very simple culture system on agar Petridishes with plant nutrients added to the agar (see Orsel et al. 2006 fordetails). Applicants will repeat these experiments in hydroponic cultureand soil pots to determine and compare NUE between wild types and linesover-expressing OsNRT2.3b. ¹⁵N-enriched nitrate will be used in Petridish and hydroponic experiments to measure and compare nitrate influxrates between wild types and overexpressing lines (see Orsel et al. 2006for methods). Plants will be grown and compared in mixed nitrogensupplies, that include ammonium nitrate or nitrate as the only nitrogensource.

3. Expression of OsNRT2.3b in Tobacco

Method and Materials:

Over-expression Vector Construction and Transgenic Plants

The open reading frames of OsNRT2.3b were amplified by gene specificprimers (Table 1). The fragment was treated with restriction enzymes,inserted into vectors and sequenced before transformation. Nicotianatabacum cultivar 89 embryonic calli were transformed usingAgrobacterium-mediated methods (Ai et al. 2009. One copy insertion T0plants were harvested and grown to generate T1 plants (FIG. 1).Homozygous T1 plants were taken for T2 production.

Southern-Blot

The independent transgenic lines with gene knockdown of OsNRT2.3a,namely r1 and r2, were determined by Southern-blot analysis followingthe procedures described previously (Jia et al., 2011).

Semi-quantitative RT-PCR

Total RNA was isolated from 100 mg of plant material with Trizol reagent(Invitrogen, Carlsbad, Calif., USA). Total RNA concentrations weredetermined by UV spectrophotometry (Eppendorf, Biophotometer, Germany) 2μg of total RNA from each sample was used as template for thefirst-strand cDNA synthesis, which was performed using M-MLV reversetranscriptase (Fermentas, Foster City, Calif., USA) according to themanufacturer's manual. The PCR amplification was performed using Taq DNApolymerase (Fermentas, Foster City, Calif., USA) for target genes withspecific primers shown below.

4. Expression of OsNRT2.3b in Wheat

The phloem localised expression of NRT2.3b, and recent findings thatsignificant amounts of nitrate are transported in the phloem e.g. Fan etal. 2009 (previously it was generally assumed that nitrate istransported from the root to the shoot in the xylem), together with theimportant role of the phloem in pH homeostasis suggest that phloemspecific expression of OsNRT2.3b may be important for the resultsreported (e.g. improved NUE). For these reasons, Applicants used bothubiquitin and a phloem-specific promoters to drive expression ofOsNRT2.3b in wheat. The ubiquitin promoter was used for thetransformation as shown in FIGS. 27 and 28. The construction of35S-OsNRT2.3b vector was described in rice transformation and wheat wereproduced by particle bombardment of calli cultured from immature embryosof susceptible variety Yangmai 158 as described (by Cao et al). Thetransgenic plant showed increased yield compared to wt plants, see FIGS.27 and 28.

5. Pathogen Resistance of Transgenic Rice

Transgenic rice plants expressing OsNRT2.3b generated as described abovewere analysed in field trails in Hainan for pathogen resistance. Themain rice diseases in Hainan, Fusarium wilt, Leaf blight and Striperust. For each plot, the survival rates were counted by the rice plantsnumber at harvest/rice plants transferred at the beginning of January.Transgenic plants showed better survival rates compared to wt plants(FIG. 26).

The Primers Used for RT-PCR of OsNRT2.3b Gene

Genes Primers

OsNRT2.3b (AK072215) F: (SEQ ID No. 21) 5′-CGTTCGCCGTGTT-3′ R:(SEQ ID No. 22) 5′-TCGAAGCGGTCGTAGAAG-3′ Actin F: (SEQ ID No. 23)5′-TTATGGTTGGGATGGGACA-3′ R: (SEQ ID No. 24) 5′-AGCACGGCTTGAATAGCG-3′The Primers Used for Over-expression Constructs

PROMOTER VECTOR PRIMERS ENZYMES CaMV-35S pCAMBIA1302 FatCCATGGAGATCTCAGGGCACAGCGGATG Bgl II (SEQ ID No. 25) RatCCATGGAGATCT ACACCCCGGCCGG Bgl II (SEQ ID No. 26) Ubiquitin pTCK303 FcaACTAGTGCTACCACGTGTTGGAGATG SpeI (SEQ ID No. 27) RGaACTAGTGAGCAAACCACCAACAAGC SpeI (SEQ ID No. 28)The Primers Used for Subcloning of OsNRT2 Genes and OsNAR2 Genes intopT7Ts

Plasmid linearization Promoter for Gene Clone vector Subcloning primerssites RNA synthesis OsNRT2.3b pLambda-FLC I F: AATCAGATCTTTGGAGCTCCACCXba I T7 (AK072215) GC (SEQ ID No. 29) R: CAGAACTAGTCCCCCCCTCGAAGG (SEQ ID No. 30)The Primers Used for H167R Site Mutant of OsNRT2.3b

Mutation Mutagenic primer Codon change New restriction site H167R-FGCCATT CGA AAGATCGGTAGCACGC (SEQ ID No. 31) CAC(H)-CGA(R)Csp45 I (TTCGAA) (original sequence: GCCATC CAC AAGATC GGTAGCACGC)^(a)(SEQ ID No. 32) H167R-R GCATTCTAGATTCGAATGGCCTCGTACACG Xba I (TCTAGA)(SEQ ID No. 33) H167RB- T7 XbaI (TCTAGA) Csp45I (TTCGAA) F67RB-RGCATTCTAGATT CGA A TGGCCTCGTACACG (SEQ ED No. 34) ^(a)The product of ATTand ATC is the same amino acid, isoleucine.The Primers Used for RT-PCR of OsNRT2.3 Gene

Genes Primers OsNRT2.3a (AK072215) F: 5′-GCTCATCCGCGACACCCT-3′(SEQ ID No. 35) R: 5′-GTCGAAGCGGTCGTAGAA-3′ (SEQ ID No. 36)OsNRT2.3b (AK072215) F: 5′-CGTTCGCCGTGTT-3′ (SEQ ID No. 37)R: 5′-TCGAAGCGGTCGTAGAAG-3′ (SEQ ID No. 38) OsActin (NM_197297)F: 5′-TTATGGTTGGGATGGGACA-3′ (SEQ ID No. 39) R: 5′-AGCACGGCTTGAATAGCG-3′(SEQ ID No. 40)

TABLE 1 The growth period differences between OsNRT2.3b over-expressionplants and Nipponbare wild type in FIG. 16 pot experiments. Date (y/m/d)sowing transplanting 50% heading flowering maturity WT 2011 May 10 2011Jun. 10 2011 Aug. 16 2011 Aug. 21 2011 Oct. 8 b-S2 2011 May 10 2011 Jun.10 2011 Sep. 1 2011 Sep. 6 2011 Oct. 28 b-S6 2011 May 10 2011 Jun. 102011 Sep. 2 2011 Sep. 8 2011 Oct. 28 Note: The soil pot experiment wasperformed with ten replications in an experimental farm of NanjingAgricultural University (data shown in FIG. SF9). The acid soil (pH 6.0,soil: water = 1:1) collected from the farm. One wild type, b-S2 and b-S6plants which belong to two independent lines were grown in each potcontaining 15 kg of air-dried soil with 2.25 g N added (n = 10). Thesoil in the pot was flooded for 1 day before transplanting and the watermaintained between 5 and 10 cm deep until 15 days before harvest.Maturity was recorded when most of the panicles in plot showed completeloss of green color. Five replicate pots of plant samples were harvestedwhen WT plants were at maturity, and other five replication pots ofplant samples were harvested when b-S2 and b-S6 plants were at maturity.The plants were dug out and separated into vegetative biomass andgrains. All plant samples were oven-dried at 70° C., weighed and groundinto powder, and were then subsampled for N determinations. Nconcentration in plant tissue and seed was determined by the standardmacro-Kjeldahl procedure.

TABLE 2 The Agronomic traits of OsNRT2.3b over expression plants and WTin FIG. 16 pot experiments. Panicle Panicle Number of Number of SeedWeight/ length weight primary secondary number/ 1000 Ripening (cm) (g)rachis rachis Panicle seeds (g) Rate (%) WT 21.4 ± 0.4b 3.2 ± 0.2b 10.0± 0.5b 20.4 ± 1.6a 125.0 ± 7.9b  24.1 ± 0.4a 83.9 ± 3.3a b-S2 26.2 ±0.7a 4.5 ± 0.3a 14.2 ± 0.4a 29.8 ± 2.6a 225.3 ± 9.4a  24.6 ± 0.4a 87.0 ±2.5a b-S6 26.1 ± 0.4a 4.4 ± 0.1a 13.9 ± 0.4a 27.9 ± 3.0a 218.0 ± 11.8a24.3 ± 0.4a 87.0 ± 2.6a Note: Values are mean ±S.E (n = 10), smallletters indicate significance of difference at 5% levels with One-wayANOVA analysis

TABLE 3 The effect of OsNRT2.3b over-expression on plant biomasstransfer and accumulation in FIG. 16 pot experiments. e_(D) − (e_(D) −(e_(D) − f_(D) (f_(D) − f_(GD)) (f_(D) − f_(GD)))/ (f_(D) − f_(GD)))/Line (g/plant) (g/plant) e_(D)(%) f_(GD)(%) WT 47.7 ± 1.0b  2.1 ± 0.4b 5.9 ± 1.3b 14.3 ± 2.8b b-S2 61.0 ± 0.7a 11.0 ± 0.9a 21.9 ± 1.7a 50.2 ±3.9a b-S6 61.7 ± 0.6a 11.9 ± 0.8a 23.4 ± 1.6a 52.8 ± 3.1a Note: 1)Values are mean ± S.E (n = 10). Small letters (a, b) indicatesignificance of difference at 5% levels compared with WT; 2) The potexperiments were conducted as described in Table 1; Dry mattertranslocation was calculated as e_(D) − (f_(D) − f_(GD)); Dry mattertranslocation efficiency was calculated as (e_(D) − (f_(D) −f_(GD)))/e_(D); The contribution of dry matter translocation wascalculated as (e_(D) − (f_(D) − f_(GD)))/f_(GD).

TABLE 4 The effect of OsNRT2.3b over-expression on plant nitrogentransfer and accumulation in FIG. 18 pot experiments. e_(N) − e_(N) −e_(N) − f_(N) (f_(N) − f_(GN)) (f_(N) − f_(GN))/ (f_(N) − f_(GN))/ Line(mg/plant) (mg/plant) e_(N)(%) f_(GN)(%) WT 424.6 ± 6.4b 109.4 ± 2.7b29.5 ± 0.9b 67.2 ± 2.9b b-S2 557.7 ± 2.9a 199.0 ± 4.4a 37.7 ± 0.6a 87.0± 0.8a b-S6 556.1 ± 9.3a 209.7 ± 3.2a 39.6 ± 0.4a 88.6 ± 1.2a Note: 1)Values are mean ± S.E (n = 10). Small letters (a, b) indicatesignificance of difference at 5% levels compared with WT; 2) The potexperiments were conducted as described in Table 1; 3). Nitrogentranslocation was calculated as e_(N) − (f_(N) − f_(GN)); nitrogentranslocation efficiency was calculated as e_(N) − (f_(N) −f_(GN))/e_(N); the nitrogen translocation contribution was calculated ase_(N) − (f_(N) − f_(GN))/f_(GN);

TABLE 5 The Agronomic traits of OsNRT2.3b over expression plants and WTin FIG. 2a field experiments. 0 kg N 75 kg N 150 Kg N 300 kg N Dryweight (g/plant) WT 21.2 ± 0.6b 21.9 ± 1.1b 30.2 ± 1.0b 28.2 ± 4.0b b-U135.0 ± 2.2a 40.5 ± 2.3a 57.1 ± 2.3a 60.8 ± 5.4a b-U2 37.5 ± 1.7a 40.5 ±3.1a 59.4 ± 3.4a 64.3 ± 3.5a b-S2 37.1 ± 1.9a 40.1 ± 3.6a 57.9 ± 4.1a63.4 ± 5.7a b-S6 38.4 ± 3.1a 40.6 ± 2.8a 60.6 ± 4.8a 65.5 ± 5.2aEffective tillering No. WT  9.3 ± 0.9a  9.3 ± 0.8a 11.0 ± 1.5a 11.3 ±1.1a b-U1  8.1 ± 0.9a  8.5 ± 0.9a 10.2 ± 1.5a  9.3 ± 1.3a b-U2  8.1 ±1.1a  8.6 ± 1.0a  9.2 ± 1.8a  9.2 ± 1.2a b-S2  8.2 ± 1.2a  8.4 ± 13a 9.3 ± 1.9a  9.5 ± 1.2a b-S6  8.3 ± 1.1a  8.6 ± 1.1a  9.4 ± 1.7a  9.2 ±1.1a Seed No./panicle WT  116 ± 4.4b  119 ± 7.1b  117 ± 6.4b  120 ± 6.4bb-U1  140 ± 8.0a  159 ± 9.5a  142 ± 8.0a  154 ± 7.0a b-U2  148 ± 9.1a 164 ± 10.1a  165 ± 9.1a  167 ± 11.1a b-S2  148 ± 8.6a  160 ± 9.1a  165± 9.6a  163 ± 12.6a b-S6  152 ± 8.9a  174 ± 11.4a  170 ± 8.9a  180 ±13.9a Weight/1000 seeds WT 23.2 ± 0.2a 24.4 ± 0.2a 24.6 ± 0.2a 25.0 ±0.6a b-U1 23.2 ± 0.2a 24.2 ± 0.4a 24.2 ± 0.3a 25.0 ± 0.3a b-U2 23.0 ±0.3a 24.3 ± 0.2a 24.1 ± 0.4a 25.2 ± 0.6a b-S2 22.9 ± 0.3a 24.2 ± 0.3a24.1 ± 0.5a 25.0 ± 0.8a b-S6 23.0 ± 0.3a 24.3 ± 0.4a 24.1 ± 0.4a 25.0 ±0.8a Ripening rate (%) WT 64.9 ± 2.4b 68.1 ± 2.2b 78.9 ± 2.8b 83.3 ±2.2b b-U1 72.0 ± 2.0a 78.0 ± 1.5a 88.0 ± 2.9a 95.1 ± 3.0a b-U2 73.0 ±1.9a 78.8 ± 2.9a 88.3 ± 2.4a 94.5 ± 2.9a b-S2 72.0 ± 2.6a 78.8 ± 2.3a89.0 ± 2.2a 93.5 ± 1.6a b-S6 74.0 ± 2.8a 79.5 ± 2.4a 88.0 ± 2.6a 92.9 ±3.1a Grain weight (g/plant) WT 16.2 ± 0.4b 18.6 ± 0.9b 24.7 ± 0.8b 28.2± 1.0b b-U1 18.9 ± 1.4a 25.5 ± 1.5a 31.8 ± 1.3a 34.0 ± 2.0a b-U2 20.1 ±0.9a 27.1 ± 2.1a 32.5 ± 1.8a 36.7 ± 2.0a b-S2 20.0 ± 1.0a 25.6 ± 2.0a33.0 ± 2.3a 36.1 ± 2.3a b-S6 21.5 ± 1.7a 28.8 ± 2.0a 33.9 ± 2.7a 38.2 ±2.4a Note: Ten plants from each replication of each treatment weresampled for this agronomic analysis and three replication. Values aremean ± S.E. (n = 30). Small letters (a, b) indicate significance ofdifference at 5% levels compared with WT.

SEQUENCE LISTINGOsNRT23.b nucleic acid sequence, Accession No: AK072215 longest ORF,see http://cdna01.dna.affrc.go.jp/cDNA/report/KOME_AK072215.htmlSEQ ID No. 1 ATGGAGGCTAAGCCGGTGGCGATGGAGGTGGAGGGGGTCGAGGCGGCGGGGGGCAAGCCGCGGTTCAGGATGCCGGTGGACTCCGACCTCAAGGCGACGGAGTTCTGGCTCTTCTCCTTCGCGAGGCCACACATGGCCTCCTTCCACATGGCGTGGTTCTCCTTCTTCTGCTGCTTCGTGTCCACGTTCGCCGTGTTCGCGCGTCTGGCCATGGGCACGGCGTGCGACCTGGTCGGGCCCAGGCTGGCCTCCGCGTCTCTGATCCTCCTCACCACACCGGCGGTGTACTGCTCCTCCATCATCCAGTCCCCGTCGGGGTACCTCCTCGTGCGCTTCTTCACGGGCATCTCGCTGGCGTCGTTCGTGTCGGCGCAGTTCTGGATGAGCTCCATGTTCTCGGCCCCCAAAGTGGGGCTGGCCAACGGCGTGGCCGGCGGCTGGGGCAACCTCGGCGGCGGCGCCGTCCAGCTGCTCATGCCGCTCGTGTACGAGGCCATCCACAAGATCGGTAGCACGCCGTTCACGGCGTGGCGCATCGCCTTCTTCATCCCGGGCCTGATGCAGACGTTCTCGGCCATCGCCGTGCTGGCGTTCGGGCAGGACATGCCCGGCGGCAACTACGGGAAGCTCCACAAGACTGGCGACATGCACAAGGACAGCTTCGGCAACGTGCTGCGCCACGCCCTCACCAACTACCGCGGCTGGATCCTGGCGCTCACCTACGGCTACAGCTTCGGCGTCGAGCTCACCATCGACAACGTCGTGCACCAGTACTICTACGACCGCTTCGACGTCAACCTCCAGACCGCCGGGCTCATCGCCGCCAGCTTCGGGATGGCCAACATCATCTCCCGCCCCGGCGGCGGGCTACICTCCGACTGGCTCTCCAGCCGGTACGGCATGCGCGGCAGGCTGTGGGGGCTGTGGACTGTGCAGACCATCGGCGGCGTCCTCTGCGTGGTGCTCGGAATCGTCGACTTCTCCTTCGCCGCGTCCGTCGCCGTGATGGTGCTCTTCTCCTTCTTCGTCCAGGCCGCGTGCGGGCTCACCTTCGGCATCGTGCCGTTCGTGTCGCGGAGGTCGCTGGGGCTCATCTCCGGGATGACCGGCGGCGGGGGCAACGTGGGCGCCGTGCTGACGCAGTACATCTTCTTCCACGGCACAAAGTACAAGACGGAGACCGGGATCAAGTACATGGGGCTCATGATCATCGCGTGCACGCTGCCCGTCATGCTCATCTACTTCCCGCAGTGGGGCGGCATGCTCGTAGGCCCGAGGAAGGGGGCCACGGCGGAGGAGTACTACAGCCGGGAGTGGTCGGATCACGAGCGCGAGAAGGGTTTCAACGCGGCCAGCGTGCGGTTCGCGGAGAACAGCGTGCGCGAGGGCGGGAGGTCGTCGGCGAATGGCGGACAGCCCAGGCACACCGTCCCCGTCGACGCGTCGCCGGCCGGGGT GTGAOsNRT2.3a nucleic acid sequence, Accession No: AK109776 longest ORFSEQ ID No. 2 ATGGAGGCTAAGCCGGTGGCGATGGAGGTGGAGGGGGTCGAGGCGGCGGGGGGCAAGCCGCGGTTCAGGATGCCGGTGGACTCCGACCTCAAGGCGACGGAGTTCTGGCTCTTCTCCTTCGCGAGGCCACACATGGCCTCCTTCCACATGGCGTGGTTCTCCTTCTTCTGCTGCTTCGTGTCCACGTTCGCCGCGCCGCCGCTGCTGCCGCTCATCCGCGACACCCTCGGGCTCACGGCCACGGACATCGGCAACGCCGGGATCGCGTCCGTGTCGGGCGCCGTGTTCGCGCGTCTGGCCATGGGCACGGCGTGCGACCTGGTCGGGCCCAGGCTGGCCTCCGCGTCTCTGATCCTCCTCACCACACCGGCGGTGTACTGCTCCTCCATCATCCAGTCCCCGTCGGGGTACCTCCTCGTGCGCTTCTTCACGGGCATCTCGCTGGCGTCGTTCGTGTCGGCGCAGTTCTGGATGAGCTCCATGTTCTCGGCCCCCAAAGTGGGGCTGGCCAACGGCGTGGCCGGCGGCTGGGGCAACCTCGGCGGCGGCGCCGTCCAGCTGCTCATGCCGCTCGTGTACGAGGCCATCCACAAGATCGGTAGCACGCCGTTCACGGCGTGGCGCATCGCCTTCTTCATCCCGGGCCTGATGCAGACGTTCTCGGCCATCGCCGTGCTGGCGTTCGGGCAGGACATGCCCGGCGGCAACTACGGGAAGCTCCACAAGACTGGCGACATGCACAAGGACAGCTTCGGCAACGTGCTGCGCCACGCCCTCACCAACTACCGCGGCTGGATCCTGGCGCTCACCTACGGCTACAGCTTCGGCGTCGAGCTCACCATCGACAACGTCGTGCACCAGTACTTCTACGACCGCTTCGACGTCAACCTCCAGACCGCCGGGCTCATCGCCGCCAGCTTCGGGATGGCCAACATCATCTCCCGCCCCGGCGGCGGGCTACTCTCCGACTGGCTCTCCAGCCGGTACGGCATGCGCGGGAGGCTGTGGGGGCTGTGGACTGTGCAGACCATCGGCGGCGTCCTCTGCGTGGTGCTCGGAATCGTCGACTTCTCCTTCGCCGCGTCCGTCGCCGTGATGGTGCTCTTCTCCTTCTTCGTCCAGGCCGCGTGCGGGCTCACCTTCGGCATCGTGCCGTTCGTGTCGCGGAGGTCGCTGGGGCTCATCTCCGGGATGACCGGCGGCGGGGGCAACGTGGGCGCCGTGCTGACGCAGTACATCTTCTTCCACGGCACAAAGTACAAGACGGAGACCGGGATCAAGTACATGGGGCTCATGATCATCGCGTGCACGCTGCCCGTCATGCTCATCTACTTCCCGCAGtGGGGCGGCATGCTCGTAGGCCCGAGGAAGGGGGCCACGGCGGAGGAGTACTACAGCCGGGAGTGGTCGGATCACGAGCGCGAGAAGGGTTTCAACGCGGCCAGCGTGCGGTTCGCGGAGAACAGCGTGCGCGAGGGCGGGAGGTCGTCGGCGAATGGCGGACAGCCCAGGCACACCGTCCCCGTCGACGCGTCGCCGGCCGGGGTGTGAOsNRT2.3b amino acid sequence (Longest ORF) SEQ ID No. 3MEAKPVAMEVEGVEAAGGKPRFRMPVDSDLKATEFWLFSFARPHMASFHMAWFSFFCCFVSTFAVFARLAMGTACDLVGPRLASASLILLTTPAVYCSSIIQSPSGYLLVRFFTGISLASFVSAQFWMSSMFSAPKVGLANGVAGGWGNLGGGAVQLLMPLVYEAIHKIGSTPFTAWRIAFFIPGLMQTFSAIAVLAFGQDMPGGNYGKLHKIGDMHKDSFGNVLRHALTNYRGWILALTYGYSFGVELTIDNVVHQYFYDRFDVNLQTAGLIAASFGMANIISRPGGGLLSDWLSSRYGMRGRLWGLWTVQTIFFVLCVVLGIVDFSFAASVAVMVLFSFFVQAACGLTFGIVPFVSRRSLGLISGMTGGGGNVGAVLTQYIFFHGTKYKTETGIKYMGLMIIACTLPVMLIYFPQWGGMLVGPRKGATAEEYYSREWSDHEREKGFNAASVRFAENSVREGGRSSANGGQPRHTVPVD ASPAGVOsNRT2.3a amino acid sequence SEQ ID No. 4MEAKPVAMEVEGVEAAGGKPRFRMPVDSDLKATEFWLFSFARPHMASFHMAWFSFFCCFVSTFAAPPLLPLIRDTLGLTATDIGNAGIASVSGAVFARLAMGTACDLVGPRLASASLILLTTPAVYCSSIIQSPSGYLLVRFFTGISLASFVSAQFWMSSMFSAPKVGLANGVAGGWGNLGGGAVQLLMPLVYEAIHKIGSTPFTAWRIAFFIPGLMQTFSAIAVLAFGQDMPGGNYGKLHKTGDMHKDSFGNVLRHALTNYRGWILALTYGYSFGVELTIDNVVGQYFYDRFDVNLQTAGLIAASFGMANIISRPGGGLLSDWLSSRYGMRGRLWGLWTVQTIGGVLCVVLGIVDFSFAASVAVMVLFSFFVQAACGLTFGIVPFVSRRSLGLISGMTGGGGNVGAVLTQYIFFHGTKYKTETGIKYMGLMIIACTLPVMLIYFPQWGGMLVGPRKGATAEEYYSREWSDHEREKGFNAASVRFAENSVREGGRSSANGGQPRHTVPVDASPAGV Phloem promoter sequenceSEQ ID No. 5 61 CAAATGTGCA ATGCTGATTA GAGTTTGCAG ATGCTGTTTG GTTTAGTTTAGATGTGGCATTTTGTTAGTG GTTTCTTTGA TGAAAAATTC TTGGCTATGA TAAAGTTTGCTTTCTGAATATATGAATAGT GGCCATGGTT CAAGAAACTC CAGTTAGGTG GGATAATTTATGGTGATTCTGGGCGCAATT CGGGGAAATT TTTTTTGGCG AGAATCTTAT CATTGAGATAAAGAGGGCAAGAATATCAAC AGACTTTTAA TCTTAATAAA AAGCACTCTT AGCGTAAGAGCAAAGCATTGCAATCTCGTG TGACAAGAAC GTTTCTTTTT CTCCATCTTT TTCTTTTTTACCAAAAAATGAGTGTTGCCA ACTGCTGCAC CTTCTTAGGC CGGTTTGTTC TTGTTTGGAACGCACGGAATGCCCGATGCA AAAAAAAAAA AGAAATGCTG TTAACAAATC ACTGTCCTGACACGGCTAATTAGGTGGTAA TTTGGTGCAT CTGCAAAGAA GCAACAGATG CTTTCTTTCACTGAAAGCATATTTGCATGA TTTCTTGTTT CTGCTTGTCC TCTCTCTGAT GCTGACTGTATTCCACTCTGCGCTGTAATG CCATGTTAGT GATTAATATG TTCAAAAGAG CATAAAAGAATTGCCAATTGGATGTTAGAG ATTACTGTGT TGTTCAAAAG AGCATAAAAG AATTACCAATTTGATGGTAGATGTTACTAG CACCACCTTG GTGTTTGCCC ATGGTTTTCT GCAATTCTGCCCATGATCTTTCTGCTTTTC TGAAAGACCT ATGTTTCAGA GGTCAAGCTT CTGGAAGGTTATTAGGAGGGATGAGTCGTC ATTTTGTCTG TGGGCCCCAC TAGTCAGTGT CAATAGTTGTAAAGGGTAGAAATTTTCTTG CTGTTTTTCT TGGAAACAAT TTCATTGCGC CTGATCTGATGGTCGGTCTGGTAATCAAAT CACCAGATCC TGAAATCCAC CAAATCAAAC CGTGAGATTTTTGCAGAGGCAAAACAAGAA AAGCATCTGC TTTATTTCTC TCTTGCTTTC TTTTCATCCCCAACCAGTCCTTTTTTCTTC TGTTTATTTG TAGAAGTCTA CCACCTGCAG TCTATTATTCTACAGAGAAAAAGATTGAAG CTTTTTTTCT CCAAAGCTGA CAATGGTGCC GGCATATGCTAATAGGATACTCCCTTCGTC TAGGAAAAAA CCAACCCACT ACAATTTTGA ATATATATTTATTCAGATTTGTTATGCTTC CTACTCCTTC TCAGGTATGG TGAGATATTT CATAGTATAATGAATTTGGACATATATTTG TCCAAATTCA TCGCATTATG AAATGTCTCG TTCGATCTATGTTGTTATATTATAGACGGA GATAGTAGAT TCGGTTATTT TTGGACAGAG AAAGTACTCGCCTGTGCTAGTGACATGATT AGTGACACCA TCAGATTAAA AAAACATATG TTTTGATTAAAAAAATGGGGAATTTGGGGG GAGCAATAAT TTGGGGTTAT CCATTGCTGT TTCATCATGTCAGCTGAAAGGCCCTACCAC TAAACCAATA TCTGTACTAT TCTACCACCT ATCAGAATTCAGAGCACTGGGGTTTTGCAA CTATTTATTG GTCCTTCTGG ATCTCGGAGA AACCCTCCATTCGTTTGCTCTTAATTAAAA GGGCAATTCT GCAGATATCC ATCACACTGG CGGCCGCTCGAGCATGCATCTAGAGGCCCA ATTCGCCCAZea mays Id No. GRMZM2G455124* nucleic acid sequence SEQ ID No. 6ATGGCGGAGGGGGAGTTCAAGCCCGCGGCGATGCAGGTGGAGGCTCCTGCCGAGGCGGCGGCGGCGCCGTCCAAGCCGCGGTTCAGGATGCCCGTCGACTCCGACAACAAGGCCACCGAGTTCTGGCTCTTCTCCTTCGCGAGGCCGCACATGAGCGCCTTCCACATGTCGTGGTTCTCCTTCTTCTGCTGCTTCCTCTCCACCTTCGCGGCGCCGCCGCTGCTCCCGCTCATCCGGGACACGCTGGGGCTCACGGCCACGGACATCGGCAACGCCGGGATCGCCTCCGTGTCCGGCGCGGTCTTCGCGCGCGTGGCCATGGGCACGGCGTGCGACCTGGTGGGCCCGCGCCTGGCGTCCGCGGCCATCATACTCCTCACCACGCCCGCCGTCTACTACTCCGCCGTCATCGACTCCGCCTCGTCCTACCTGCTCGTGCGCTTCTTCACGGGCTTCTCGCTCGCGTCCrTCGTGTCCACGCAGTTCTGGATGAGCTCCATGTTCTCGCCGCCCAAGGTGGGGCTGGCCAACGGCGTCGCCGGGGGGTGGGGCAACCTCGGCGGCGGCGCCGTGCAGCTCATCATGCCGCTCGTGTTCGAGGCCATCCGCAAGGCCGGGGCCACGCCGTTCACGGCGTGGCGCGTCGCCTTCTTCGTCCCGGGCCTGCTGCAGACGCTGTCGGCCGTCGCCGTGCTGGCGTTCGGCCAGGACATGCCCGACGGCAACTACCGCAAGCTGCACAGGTCCGGCGACATGCACAAGGACAGCTTCGGCAACGTGCTCCGCCACGCCGTCACCAACTACCGCGCCTGGATCCTGGCGCTCACCTACGGATACTGCTTCGGCGTGGAGCTCGCCGTGGACAACATCGTCGCGCAGTACTTCTACGACCGCTTCGGCGTCAAGCTCAGCACCGCCGGCTTCATCGCCGCCAGCTTCGGGATGGCCAACATCGTCTCCCGCCCCGGCGGCGGCCTCCTGTCGGACTGGCTCTCCAGCCGCTTCGGCATGCGCGGCAGGCTGTGGGGCCIGTGGGTGGTGCAGACCATCGGGGGCGTCCTCTGCGTCGTGCTCGGCGCCGTCGACTACTCCTTCGCCGCGTCCGTGGCCGTCATGATACTCTTCTCCATGTTCGTGCAGGGGGCCTGCGGGCTCACCTTTGGCATCGTCCCGTTCGTCTCCCGAAGGTCGCTGGGGCTCATCTCCGGCATGACCGGCGGCGGCGGCAACGTGGGCGCCGTGCTCACGCAGCTCATCTTCTTCCACGGATCCAAGTACAAGACGGAGACGGGGATCAAGTACATGGGGTTCATGATCATCGCCTGCACGTTGCCCATCACGCTCATCTACTTCCCGCAGTGGGGCGGCATGTTCCTGGGGCCGCGGCCCGGGGCGACGGCGGAGGACTACTACAACCGGGAGTGGACAGCGCACGAGTGCGACAAGGGTTTCAACACCGCGAGCGTACGCTTTGCGGAGAACAGCGTGCGGGAAGGGGGACGCTCGGGCAGCCAGTCCAAGCACACTACTGTGCCCGTCGAGTCCTCGCCGGCCGACGTG TGAZea mays Id No. GRMZM2G455124* amino acid sequence SEQ ID No. 7MAEGEFKPAAMQVEAPAEAAAAPSKPRFRMPVDSDNKATEFWLFSFARPHMSAFHMSWFSFFCCFLSTFAAPPLLPLIRDTLGLTATDIGNAGIASVSGAVFARVAMGTACDLVGPRLASAAIILLTTPAVYYSAVIDSASSYLLVRFFTGFSLASFVSTQFWMSSMFSPPKVGLANGVAGGWGNLGGGAVQLIMPLVFEAIRKAGATPFTAWRVAFFVPGLLQTLSAVAVLAFGQDMPDGNYRKLHRSGDMHKDSFGNVLRHAVTNYRAWILALTYGYCFGVELAVDNIVAQYFYDRFGVKLSTAGFIAASFGMANIVSRPGGGLLSDWLSSRFGMRGRLWGLWVVQTGGVLCVVLGAVDYSFAASVAVMILFSMFVQAACGLTFGIVPFVSRRSLGLISGMTGGGGNVGAVLTQLIFFHGSKYKTETGIKYMGFMIIACILPITLIYFPQWGGMFLGPRPGATAEDYYNREWTAHECDKGFNTASVRFAENSVREGGRSGSQSKHTTVPVESSPADVGlycine max Id No. Glyma13g39850 nucleic acid sequence SEQ ID No. 8TCACACTTTCTTCCTTAATTTTCTAGCTCTTGCTACGTACTTGAATTCAATTAGTTATTAATGGCTGAGATTGAGGGTTCTCCCGGAAGCTCCATGCATGGAGTAACAGGAAGAGAACAAACATTTGTAGCCTCAGTTGCTTCTCCAATTGTCCCTACAGACACCACAGCCAAATTTGCTCTCCCAGTGGATTCAGAACACAAGGCCAAGGTTTTCAAACTCTTCTCCCTGGCCAATCCCCACATGAGAACCTTCCACCTTTCTTGGATCTCCTTCTTCACCTGCTTCGTCTCGACATTCGCAGCAGCACCTCTTGTGCCCATCATCCGCGACAACCTTAACCTCACCAAAAGCGACATTGGAAACGCCGGGGTTGCTTCTGTCTCCGGAAGCATCTTCTCAAGGCTCGCAATGGGTGCAGTCTGTGACATGTTGGGTCCACGCTATGGCTGCGCCTTCCTCATCATGCTTTCGGCCCCTACGGTGTTCTGCATGTCCTTTGTGAAAGATGCTGCGGGGTACATAGCGGTTCGGTTCTTGATTGGGTTCTCGTTGGCGACGTTTGTGTCGTGCCAGTACTGGATGAGCACGATGTTCAACAGTAAGATTATAGGGCTTGCGAATGGGACTGCTGCGGGGTGGGGGAACATGGGTGGTGGAGCCACTCAGCTCATAATGCCTTTGGTGTATGAGCTTATCAGAAGAGCTGGGGCTACTCCCTTCACTGCTTGGAGGATTGCCTTCTTTGTTCCGGGTTTCATGCATGTCATCATGGGGATTCTTGTCCTCACTCTAGGCCAGGACTTGCCTGATGGAAACCTCGGGGCCTTGCGGAAGAAGGGTGATGTAGCTAAAGACAAGTTTTCCAAGGTGCTATGGTATGCCATAACAAATTACAGGACATGGATTTTTGCTCTCCTCTATGGGTACTCCATGGGAGTTGAATTAACAACTGACAATGTCATTGCTGAGTATTTCTATGACAGATTTAATCTCAAGCTACACACTGCTGGAATCATTGCTGCTTCATTTGGAATGGCAAACTTAGTTGCTCGACCTTTTGGTGGATATGCTTCAGATGTTGCAGCCAGGCTGTTTGGCATGAGGGGAAGACTCTGGACCCTTTGGATCCTCCAAACCTTAGGAGGGGTTTTCTGTATrTGGCTTGGCCGTGCCAATTCTCTTCCTATTGCTGTATTGGCCATGATCCTGTTCTCTATAGGAGCTCAAGCTGCATGTGGTGCAACTTTTGGCATCATTCCTTTCATCTCAAGAAGGTCTTTGGGGATCATATCAGGTCTAACTGGTGCAGGTGGAAACTTTGGGTCTGGCCTCACCCAATTGGTCTTCTTTTCAACCTCCAAATTCTCTACTGCCACAGGTCTCTCCTTGATGGGTGTAATGATAGTGGCTTGCACTCTACCAGTGAGTGTTGTTCACTTCCCACAGTGGGGTAGCATGTTTCTACCACCCTCAAAAGATGTCAGCAAATCCACTGAAGAATTCTATTACACCTCTGAATGGAATGAGGAAGAGAAGCAGAAGGGTTTGCACCAGCAAAGTCTCAAATTTGCTGAGAATAGCCGATCTGAGAGAGGAAAGCGAGTGGCTTCAGCACCAACACCTCCAAATGCAACTCCCACTCATGTCTAGCCATAGCACTTCAATCAAAGAAGATCATGAAACATAATTACTGAGCAGTATTGGGAATGAAGAACCATGAGTTGAAGAATTTTCTAATAAGAAATCTTGTAACATGTAGACATAGAATGTTCTGGTTCTGGTTTGCGTGTGGTGTAAGAGTTGTCTACTTGTGGTAAGTCATAAGTATCATAATCAGTATGTCAATGCAGATCTTGATGCTGAGTATCAATAGTATCAAAAAAAAAAGlycine max Id No. Glyma13g39850 amino acid sequence SEQ ID No. 9MAEIEGSPGSSMHGVTGREQTFVASVASPIVPTDTTAKFALPVDSEHKAKVFKLFSLANPHMRTFHLSWISFFTCFVSTFAAAPLVPIIRDNLNLTKSDIGNAGVASVSGSIFSRLMAGAVCDMLGPRYGCAFLIMLSAPTVFCMSFVKDAAGYIAVRFLIGFSLATFVSCQYWMSTMFNSKIIGLANGTAAGWGNMGGGATQLIMPLVYELIRRAGATPFTAWRIAFFVPGFMHVIMGILVLTLGQDLPDGNLGALRKKGDVAKDKFSKVLWYAITNYRTWIFALLYGYSMGVELTTDNVIAEYFYDRFNLKLHTAGIIAASFGMANLVARPFGGYASDVAARLFGMRGRLWTLWILQTLGGVFCIWLGRANSLPIAVLAMILFSIGAQAACGATFGIIPFISRRSLGIISGLTGAGGNFGSGLTQLVFFSTSFFSTATGLSLMGVMIVACTLPVSVVHFPQWGSMFLPPSKDVSKSTEEFYYTSEWNEEEKQKGLHQQSLKFAENSRSERGKRVASAPTPPNATPTHVGlycine max Id No. Glyma12g30050 nucleic acid sequence SEQ ID No. 10atggctgaga ttgagggttc tcctggaagc tccatgcatg gagtaacagg aagagaacaaacattcgtag cctcaattgc ttctccaatt gtccccacag acaccacagc caaatttgctctcccagtag actcagagca caaggccaag attttcaaac tcttctccat ggccaatccccacatgagaa ccttccacct ttcttggatc tccttcttca cctgcttcgt ctcgaccttcgcagcagccc ctcttgtccc catcatccgc gacaacctta acctcaccaa aagcgacattggaaacgccg gggttgcttc tgtctccgga agcatcttct ctaggcttgc aatgggtgcggtctgtgacc tattaggtcc acgttatggc tgtgccttcc tcatcatgct ctcggccccaaccgtgttct gcatgtcctt tgtgaaagat gctgcggggt acataatggt tcggttattgatagggttct ccttggcaac gttcgtgtca tgccagtact ggatgagcac gatgttcaacagtaagatta tagggcttgc gaatggaact gctgcggggt gggggaacat gggtggtggagccactcagc tcataatgcc tttggtgtat gagcttatca gaagagctgg ggctactcccttcactgctt ggaggatagc cttctttgta ccgggtttca tgcatgtcat catggggatccttgtcctaa ctctaggcca ggacttgcct gatggaaacc ttgcggcctt gcagaagaagggtgatgtag caaaagacaa gttttccaag gtgctatggt atgccataac aaattacaggacatggattt ttgccctcct ctatgggtac tcaatgggag ttgaattgac aactgacaatgtcattgctg agtatttcta tgacaggttt aatctgaagc tgcacactgc tggaatcattgctgcttcat ttggaatggc aaacttagtt gctcgaccct ttggtggata tgcttctgatgttgcagcca gattgtttgg catgagggga agactctgga ccctttggat cctccaaacattaggagggg ttttctgtat ttggcttggc cgagccaatt ctcttcctat tgctattttggctatgatcc tgttctcttt aggagctcaa gctgcatgtg gtgcaacttt tggcatcattcccttcatct caagaaggtc attggggatc atatcaggtc tcactggtgc aggtgggaactttgggtctg gcctcaccca attggtcttc ttttcaacat ccaaattctc cactgccacaggtctctcct tgatgggtgt gatgatagtg gcttgcactc ttcctgtgag tgttgttcattttccacagt ggggtagcat gttcctacca ccatcaaaag atgtcaacaa atccactgaagaattctatt acacctctga atggaatgag gaagagaggc agaaaggctt gcatcagcaaagtctcaagt ttgctgagaa tagccgatcc gagagaggaa agcgagtggc ttcagcacca acacctccgaatgcaactcc cactcatgtcGlycine max Id No. Glyma12g300500 amino acid sequence SEQ ID No. 11MAEIEGSPGS SMHGVTGREQ TFVASIASPI VPTDTTAKFA LPVDSEHKAK IFKLFSMANPHMRTFHLSWI SFFTCFVSTF AAAPLVPIIR DNLNLTKSDI GNASVASVSG SIFSRLAMGAVCDLLGPRYG CAFLIMLSAP TVFCMSFVKD AAGYIMVRFL IGFSLATFVS CQYWMSTMFNSKIIGLANGT AAGWGNMGGG ATQLIMPLVY ELIRRAGATP FTAWRIAFFV PGFMHVIMGILVLTLGQDLP DGNLAALQKK GDVAKDKFSK VLWYAIINYR TWIGALLYGY SMGVELIIDNVIAEYFYDRF NLKLHIAGII AASFGMANLV ARPFGGYASD VAARLFGMRG RLWTLWILQTLGGVFCIWLG RANSLPIAIL AMILFSLGAQ AACGATFGII PFISRRSLGI ISGLTGAGGNFGSGLTQLVF FSTSKFSTAT GLSLMGVMIV ACTLPVSVVH FPQWGSMFLP PSKDVNKSTEEFYYTSEWNE EERQKGLHQQ SLKFAFNSRS ERGKRVASAP TPPNATPTHVHordeum vulgare Id No. MLOC_75087.1 nucleic acid sequence SEQ ID No. 12CCACGCGTCCGCTCATIGCATACGAGGTTGCCAACACTAraCAGGTGTAGCAGCAGCCAAGGCAGCTGGTGAGATGGAGGGGGAGTCCAAGCCGGCGGCGATGGGGGTGCAGGCGGCGCCCAAGGGCAAGTTCAGGATACCGGTGGACTCGGACAACAAGGCCACCGAGTTCTGGCTTTTCTCGTTCGTGAGGCCGCACATGAGCGCCTTCCACCTCTCGTGGTTCTCCTTCTTCTGCTGCTTCGTCTCCACCTTCGCCGCGCCGCCCCTCCTGCCGCTCATCCGGGACAACCTCGGCCTCACGGGCAAGGACATCGGCAACGCCGGCATCGCGTCCGTGTCCGGCGCCGTGTTCGCGCGTCTCGCCATGGGCACGGCCTGCGACCTGGTCGGGCCCCGCCTGGCGTCCGCGGCCATCATACTGCTCACCACCCCCGCGGTGTACTGCTCCGCCATCATCGAGTCCGCCTCGTCGTTCCTGCTCGTGCGCTTCTTCACGGGCITCTCGCTCGCCTCCrTCGTGTCGACGCAGTTCTGGATGAGCTCCATGTTCTCTTCGCCCAAGGTGGGGCTGGCCAATGGCGTCGCCGGCGGCTGGGGCAACCTGGGCGGGGGCGCCGTGCAGCTCCTCATGCCGCTCGTGTTCGAGGCCGTCCGCAAGATCGGCAGCACGGATTTCATCGCGTGGCGCGTCGCCTTCTTCATCCCGGGCGTCATGCAGACGTTCTCGGCCATCGCCGTGCTGGCGTTCGGGCAGGACATGCCGGACGGCAACTACCGTAAGCTGCACAAGAGCGGGGAGATGCACAAGGACAGCTTCGGCAACGTGCTGCGCCACGCGGTCACGAACTACCGCGCCTGGATCCTGGCGCTCACCTACGGCTACTCCTTCGGCGTGGAGCTCGCCGTGGACAACATCGTCGCGCAGTACTTCTACGACCGCTTCGACGTCAACCTCCACACGGCCGGGCTCATCGCCGCCAGCTTCGGGATGGCCAACATCATCTCCCGCCCGGGCGGCGGGCTCATGTCCGACTGGCTCTCCGACCGGTTCGGCATGCGCGGCAGGCTGTGGGGGCTGTGGGTCGTGCAGACCATCGGCGGCATCCTCTGCATCGTGCTCGGCATCGTCGACTACTCGTTCGGCGCGTCGGTGGCCGTCATGATCCTCTTCTCCTTCTTCGTGCAGGCGGCGTGCGGGCTCACGTTCGGCATCGTGCCGTTCGTGTCGCGGAGGTCGCTGGGGCTCATCTCCGGAATGACCGGCGGCGGCGGCAACGTGGGGGCCGTGCTGACGCAGGTCATCTTCTTCCGCGGCACCAAGTACAAGACGGAGACGGGGATCATGTACATGGGGCTGATGATCCTGGCATGCACGCTGCCCATCACGCTCATCTACTTCCCGCAGTGGGGCGGCATGTTCGTCGGGCCGCGGAAAGGGGCGACGGCGGAGGAGTACTACAGCAAGGAGTGGACCGAGGAGGAGCGTGCCAAGGGGTACAGCGCCGCGACCGAGCGTTTCGCGGAGAACAGCGTGCGCGAGGGCGGGCGGAGGGCGGCGTCGGGCAGCCAGTCAAGGCACACCGTCCCCGTCGACGGCTCGCCGGCCGACGTGTGAGGTCCGAAGAGCTCCCCGTACTACGTGGTCCACGGGTGCAATGGGGGAATACGATCGCGTCGCACGGCCGCCCGGGTTTGGGCCGTCTTCCGTGCACATACGTAGTACTACGAACGCACGCACGCACGCCGGCTTTGTGCTGCTTCTAGTACTGTACGTACGTTTGGGTTTGGTGTGCTCGCTTACCTTAATACTGCTCCGCATGTTGATGTTTATATGCTCCCTTGTGAAATACAGTTTTAAAAAAAAAAAAAAAAHordeum vulgare Id No. MLOC_75087.1 nucleic acid sequence SEQ ID No. 13MEGESKPAAMGVQAAPKGKFRIPVDSDNKATEFWLFSFVRPHMSAFHLSWFSFFCCFVSTFAAPPLLPLIRDNLGLTGKDIGNAGIASVSGAVFARLAMGTACDLVGPRLASAAIILLTTPAVYCSAIIESASSFLLVRFFTGFSLASFVSTQFWMSSMFSSPKVGLANGVAGGWGNLGGGAVQLLMPLVFEAVRKIGSTDFIAWRVAFFIPGVMQTFSAIAVLAFGQDMPDGNYRKLHKSGEMHKDSFGNVLRHAVTNYRAWILALTYGYSFGVELAVDNIVAQYFYDRFDNVLHTAGLIAASFGMANIISRPGGGLMSDWLSDRFGMRGRLWGLWVVQTIGGILCIVLGIVDYSFGASVAVMILFSFFVQAACGLTFGIVPFVSRRSLGLISGMTGGGGNVGAVLTQVIFFRGTKYKTETGIMYMGLMILACTLPITLIYFPQWGGMFVGPRKGATAEEYYSKEWTEEERAKGYSAATERFAENSVREGGRRAASGSQSRHTVPVDGSPADVBrachypodium distachyon Id No. Bradi2g47640 nucleic acid sequenceSEQ ID No. 14 ATGGQGGQGGAGTCGAAGCCGGCGGCGATGGATGTGGAGGCGCCGTCCAAGGCCAAGTTCAGGATCCCCGTGGACTCCGACAACAAGGCGACGGAGTTCTGGCTCTTCTCCTTCGCGCGGCCGCACATGAGCGCGTTCCACCTGTCGTGGCTCCTTCTTCTGCTGCTTCGTGTCCACCTTCGCGGCGCCGCCGCTGCTGCCGCTCATCCGGGACAATCTGGGGCTCACGGCCAAGGACATCGGCAACGCCGGGATCGCGTCGGTGTCGGGCGCCGTGTTCGCGCGTCTCGCCATGGGCACGGCCTGCGACCTGGTCGGCCCCCGCCTGGCGTCCGCGGCCATCATACTGCTCACCACCCCGGCGGTGTACTGCTCGGCCATCATCGACTCGGCGTCGTCGTTCCTGCTCGTGCGCTTCTTCACGGGCTTCTCCCTGGCCTCCTTCGTGTCCACGCAGTTCTGGATGAGCTCCATGTTCTCCTCGCCCAAGGTGGGTCTGGCCAACGGCGTGGCCGGGGGCTGGGGCAACCTCGGCGGCGGCGCCGTGCAGCTGATCATGCCGCTGGTGTTCGAGGTCGTGCGCAGATCGGGAGCACGCGGTTCACGGCGTGGCGCGTGGCCTTCTTCATCCCGGGCGTCATGCAGACGTTCTCGGCCATCGCCGTGCTGGCGTTCGGGCAGGACATGCCGGACGGCAACTAGCACAAGCTGCACAAGACCGGGGAGATGCACAGGGACAGCTTCCGCAACGTGCTGCGCCACGCGGTCACCAACTACCGCGCCTGGATCCTGGCGCTCACCTACGGCTACTGCTTCGGCGTGGAGCTCGCCGTGGACAACATCGTGGCGCAGTACTTCTACGACCGCTTCGGCGTCAACCTCCACACGGCGGGGCTCATCGCCGCCAGCTTCGGGATGGCCAACATCGTCTCGCGCCCGGGCGGCGGGCTCATGTCCGACTGGCTCTCGGCCCGGTTCGGCATGCGCGGCAGGCTGTGGGGCCTGTGGGTCGTGCAGACCATCGGCGGCGTCCTCTGCGTGGTGCTCGGCGTGGTGGACTACTCCTTCGGCGCGTCCGTGQCAGTCATGATACTCTTCTCCCTGTTCGTGCAGGCCGCGTGCGGGCTCACCTTCGGCATCGTGCCGTTCGTGTCGCGGAGGTCGCTGGGGCTCATCTCTGGCATGACCGGCGGCGGGGGAAATGTGGGCGCCGTGCTGACGCAGGTCATCTTCTTCCACGGGTCCAGGTACAAGACGGAGACGGGGATCATGTACATGGGGGTCATGATCATCGCGTGCACGCTGCCCATCACGCTCATCTACTTCCCGCAGTGGGGCGGCATGTTCACCGGGCCGCGGCCGGGGGCCACGGCGGAGGAGTATTACAGCTCGGAGTOGACCGAGGAGGAGCGGAAGAAAGGGTACAACGCCGCGACAGAGCGTTTCGCGGAGAACAGCCTGCGCGAGGGAGGGCGGAGGGCCGCGTCGGGCAGCCAGTCCAAGCATACCGTCCCCGTGGACGGATCACCGCCGGCCGACGTGTGAAGAAAATCCCATAGACCATAGTGTACGTTTCGTATGTCTCGCGTCTATAACGAGTCATACGGTCGCCACGGTCGCCGGTCTGGTTACGTGCGTTGGCTTTTTrATGTGTTGTACCTTTTGGCTTTTGGTGCTCCTTTGTCTTGTTGCTGTAAAAGGTTGTCAAATACTCCACTTTTCTTTTCCGCAGACGTGAAATACTTCTGTAGGTGTACGTCACTGAAAGGAAACTGTTCATATGGCATCCACATACAAAACCATGTTTTCTTATATTGCTAGTATATTCGTTTTTCTTATTTCGACGAAACTAGCATTCCGCGTCTATTATTATTCGTAAGATACTTCCGATCGAAAABrachypodium distachyon Id No. Bradi2g47640 nucleic acid sequenceSEQ ID No. 15 MGGESKPAAMDVEAPSKAKFRIPVDSDNKATEFWLFSFARPHMSAFHLSWFSFFCCFVSTFAAPPLLPLIRDNLGLTAKDIGNAGIASVSGAVFARLAMGTACDLVGPRLASAAHLLTTPAVYCSAIIDSASSFLLVRFTGFSLASFVSTQFWMSSMFSSPKVGLANGVAGGWGNLGGGAVQLIMPLVFEVVRKIGSTRFTAWRVAFFIPGVMQTFSAIAVLAFGQDMPDGNYHKLHKTGEMHRDSFRNVLRHAVTNYRAWILALTYGYCFGVELAVDNIVAQYFYDRFGVNLHTAGLIAASFGMANILVSRPGGGLMSDWLSARFGMRGRLWGLWVVQTIGGVLCVTQVIFFHGSRYKTETGIMYMGVMILACTLPITLIYFPQWGGMFTGPRPGATAEEYYSSEWTEEERKKGYNAATERFAENSLREGGRRAASGSQSKHTVPVDGSPPADVOsNRT2.3b nucleic acid sequence, Accession No: AK072215 underlinedcharacter is longest ORF SEQ ID No. 67GAGCGCCGGCCTCCCACCGGTCGCGTAAGATCACGCCCGAAATCTTTATTCATTTTCTCTCCACCGGTTGCCCTCTCGCCGCACCCAACCATCGCGCCACGCCGCGCCGCGCTGCCGGAGCCGCGCTTTCCGCTATGCTATAAGAGCTGACGCGCAGGGCACAGCGGATGTACGTACACACAGTCACTAGCTAAGCTGCTAGCCTTGCTACCACGTGTTGGAGATGGAGGCTAAGCCGGTGGCGATGGAGGTGGAGGGGGTCGAGGCGGCGGGGGGCAAGCCGCGGTTCAGGATGCCGGTGGACTCCGACCTCAAGGCGACGGAGTTCTGGCTCTTCTCCTTCGCGAGGCCACACATGGCCTCCTTCCACATGGCGTGGTTCTCCTTCTTCTGCTGCTTCGTGTCCACGTTCGCCGTGTTCGCGCGTCTGGCCATGGGCACGGCGTGCGACCTGGTCGGGCCCAGGCTGGCCTCCGCGTCTCTGATCCTCCTCACCACACCGGCGGTGTACTGCTCCTCCATCATCCAGTCCCCGTCGGGGTACCTCCTCGTGCGCTTCTTCACGGGCATCTCGCTGGCGTCGTTCGTGTCGGCGCAGTTCTGGATGAGCTCCATGTTCTCGGCCCCCAAAGTGGGGCTGGCCAACGGCGTGGCCGGCGGCTGGGGCAACCTCGGCGGCGGCGCCGTCCAGCTGCTCATGCCGCTCGTGTACGAGGCCATCCACAAGATCGGTAGCACGCCGTTCACGGCGTGGCGCATCGCCTTCTTCATCCCGGGCCTGATGCAGACGTTCTCGGCCATCGCCGTGCTGGCGTTCGGGCAGGACATGCCCGGCGGCAACTACGGGAAGCTCCACAAGACTGGCGACATGCACAAGGACAGCTTCGGCAACGTGCTGCGCCACGCCCTCACCAACTACCGCGGCTGGATCCTGGCGCTCACCTACGGCTACAGCTTCGGCGTCGAGCTCACCATCGACAACGTCGTGCACCAGTACTTCTACGACCGCTTCGACGTCAACCTCCAGACCGCCGGGCTCATCGCCGCCAGCTTCGGGATGGCCAACATCATCTCCCGCCCCGGCGGCGGGCTACTCTCCGACTGGCTCTCCAGCCGGTACGGCATGCGCGGCAGGCTGTGGGGGCTGTGGACTGTGCAGACCATCGGCGGCGTCCTCTGCGTGGTGCTCGGAATCGTCGACTTCTCCTTCGCCGCGTCCGTCGCCGTGATGGTGCTCTTCTCCTTCTTCGTCCAGGCCGCGTGCGGGCTCACCTTCGGCATCGTGCCGTTCGTGTCGCGGAGGTCGCTGGGGCTCATCTCCGGGATGACCGGCGGCGGGGGCAACGTGGGCGCCGTGCTGACGCAGTACATCTTCTTCCACGGCACAAAGTACAAGACGGAGACCGGGATCAAGTACATGGGGCTCATGATCATCGCGTGCACGCTGCCCGTCATGCTCATCTACTTCCCGCAGTGGGGCGGCATGCTCGTAGGCCCGAGGAAGGGGGCCACGGCGGAGGAGTACTACAGCCGGGAGTGGTCGGATCACGAGCGCGAGAAGGGTTTCAACGCGGCCAGCGTGCGGTTCGCGGAGAACAGCGTGCGCGAGGGCGGGAGGTCGTCGGCGAATGGCGGACAGCCCAGGCACACCGTCCCCGTCGACGCGTCGCCGGCCGGGGTGTGAAGAATGCCACGGACAATAAGGTCGCGGTTGTAGTACAACTGTACAAATTGATGGTACGTGTCGTTTGACCGCGCGCGCGCACAGTGTGGGTCGTGGCCTCGTGGGCTTAGTGGAGTACAGTGAGGGGTGTACGTGTGTCGTGGCGCGCGCGGTCACCTCGGTGGCCTTGGGATTGGGGGGGCACTATACGCTAGTACTCCAGATATATACGGGTTTGATTTACTTCTGTGGATCGGCGCTTGTTGGTGGTTTGCTCCCTGTGGTTTTTGTGATGGTAATCATACTCATACTCAAACAGTCAAAACTTTTTGATGCGOsNRT2.3a nucleic acid sequence, Accession No: AK109776 underlinedcharacter is longest ORF SEQ ID No. 68AGTCACTAGCTAAGCTGCTAGCCTTGCTACCACGTGTTGGAGATGGAGGCTAAGCCGGTGGCGATGGAGGTGGAGGGGGTCGAGGCGGCGGGGGGCAAGCCGCGGTTCAGGATGCCGGTGGACTCCGACCTCAAGGCGACGGAGTTCTGGCTCTTCTCCTTCGCGAGGCCACACATGGCCTCCTTCCACATGGCGTGGTTCTCCTTCTTCTGCTGCTTCGTGTCCACGTTCGCCGCGCCGCCGCTGCTGCCGCTCATCCGCGACACCCTCGGGCTCACGGCCACGGACATCGGCAACGCCGGGATCGCGTCCGTGTCGGGCGCCGTGTTCGCGCGTCTGGCCATGGGCACGGCGTGCGACCTGGTCGGGCCCAGGCTGGCCTCCGCGTCTCTGATCCTCCTCACCACACCGGCGGTGTACTGCTCCTCCATCATCCAGTCCCCGTCGGGGTACCTCCTCGTGCGCTTCTTCACGGGCATCTCGCTGGCGTCGTTCGTGTCGGCGCAGTTCTGGATGAGCTCCATGTTCTCGGCCCCCAAAGTGGGGCTGGCCAACGGCGTGGCCGGCGGCTGGGGCAACCTCGGCGGCGGCGCCGTCCAGCTGCTCATGCCGCTCGTGTACGAGGCCATCCACAAGATCGGTAGCACGCCGTTCACGGCGTGGCGCATCGCCTTCTTCATCCCGGGCCTGATGCAGACGTTCTCGGCCATCGCCGTGCTGGCGTTCGGGCAGGACATGCCCGGCGGCAACTACGGGAAGCTCCACAAGACTGGCGACATGCACAAGGACAGCTTCGGCAACGTGCTGCGCCACGCCCTCACCAACTACCGCGGCTGGATCCTGGCGCTCACCTACGGCTACAGCTTCGGCGTCGAGCTCACCATCGACAACGTCGTGCACCAGTACTTCTACGACCGCTTCGACGTCAACCTCCAGACCGCCGGGCTCATCGCCGCCAGCTTCGGGATGGCCAACATCATCTCCCGCCCCGGCGGCGGGCTACTCTCCGACTGGCTCTCCAGCCGGTACGGCATGCGCGGCAGGCTGTGGGGGCTGTGGACTGTGCAGACCATCGGCGGCGTCCTCTGCGTGGTGCTCGGAATCGTCGACTTCTCCTTCGCCGCGTCCGTCGCCGTGATGGTGCTCTTCTCCTTCTTCGTCCAGGCCGCGTGCGGGCTCACCTTCGGCATCGTGCCGTTCGTGTCGCGGAGGTCGCTGGGGCTCATCTCCGGGATGACCGGCGGCGGGGGCAACGTGGGCGCCGTGCTGACGCAGTACATCTTCTTCCACGGCACAAAGTACAAGACGGAGACCGGGATCAAGTACATGGGGCTCATGATCATCGCGTGCACGCTGCCCGTCATGCTCATCTACTTCCCGCAGTGGGGCGGCATGCTCGTAGGCCCGAGGAAGGGGGCCACGGCGGAGGAGTACTACAGCCGGGAGTGGTCGGATCACGAGCGCGAGAAGGGTTTCAACGCGGCCAGCGTGCGGTTCGCGGAGAACAGCGTGCGCGAGGGCGGGAGGTCGTCGGCGAATGGCGGACAGCCCAGGCACACCGTCCCCGTCGACGCGTCGCCGGCCGGGGTGTGAAGAATGCCACGGACAATAAGGTCGCGGTTGTAGTACAACTGTACAAATTGATGGTACGTGTCGTTTGACCGCGCGCGCGCACAGTGTGGGTCGTGGCCTCGTGGGCTTAGTGGAGTACAGTGAGGGGTGTACGTGTGTCGTGGCGCGCGCGGTCACCTCGGTGGCCTTGGGATTGGGGGGGCACTATACGCTAGTACTCCAGATATATACGGGTTTGATTTACTTCTGTGGATCGGCGCTTGTTGGTGGTTTGCTCCCTGTGGTTTTTGTGATGGTAATCATACTCATACTCAAACAGTC

REFERENCES

-   1. Edgerton, M. D. Increasing crop productivity to meet global needs    for feed, food, and fuel. Plant Physiol. 149, 7-13 (2009).-   2. Ju, X. T. et al. Reducing environmental risk by improving N    management in intensive Chinese agricultural systems. Proc Natl Acad    Sci USA. 106, 3041-3046 (2009).-   3. Guo, J. H. et al. Significant acidification in major Chinese    croplands. Science 327, 1008-1010 (2010).-   4. Xu, G. H., Fan, X. R., Miller, A. J. Plant nitrogen assimilation    and use efficiency. Ann Rev Biol. 63, 153-182 (2012).-   5. Robertson, G. P. & Vitousek, P. M. Nitrogen in agriculture:    balancing the cost of an essential resource. Ann Rev Environ and    Res. 34, 97-125 (2009).-   6. Tilman, D., Balzer, C., Hill, J., Befort, B. L. Global food    demand and the sustainable intensification of agriculture. Proc Nat    Acad Sci USA. 108, 20260-20264 (2011).-   7. Sutton, M. A., Erisman, W., Leip, A., van Grinsven, H.,    Winiwarter, W. Too much of a good thing. Nature 472, 159-61 (2011).-   8. Li, Y. L., Fan, X. R., Shen, Q. R. The relationship between    rhizosphere nitrification and nitrogen use efficiency in rice    plants. Plant Cell Environ. 31, 73-85 (2008).-   9. Kirk, G. J. D. & Kronzucker, H. J. The potential for    nitrification and nitrate uptake in the rhizosphere of wetland    plants: a modeling study. Ann Bot. 96, 639-646 (2005).-   10. Zhu, Y. et al. Adaptation of plasma membrane H(+)-ATPase of rice    roots to low pH as related to ammonium nutrition. Plant Cell    Environ. 32, 1428-1440 (2009).-   11. Araki, R., Hasegawa, H. Expression of rice (Oryza sativa L.)    genes involved in high-affinity nitrate transport during the period    of nitrate induction. Breeding Sci. 56, 295-302 (2006).-   12. Cai, C. et al. Gene structure and expression of the    high-affinity nitrate transport system in rice roots. J Integ Plant    Biol. 50, 443-451 (2008).-   13. Feng, H. M. et al. Spatial expression and regulation of rice    high-affinity nitrate transporters by nitrogen and carbon status. J    Exp Bot. 62, 2319-2332 (2011).-   14. Yan, M. et al. Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2    and OsNRT2.3a nitrate transporters to provide uptake over high and    low concentration ranges. Plant Cell Environ. 34, 1360-1372 (2011).-   15. Katayama, H. et al. Production and characterization of    transgenic rice plants carrying a high-affinity nitrate transporter    gene (OsNRT2.1). Breeding Sci. 59, 237-243 (2009).-   16. Suzui, N., Nakamura, S., Fujiwara, T., Hayashi, H., Yoneyama, T.    A putative acyl-CoA-binding protein is a major phloem sap protein in    rice (Oryza sativa L.). J Exp Bot. 57, 2571-2576 (2006).-   17. Rao, T. P., Yano, K., Iijima, M., Yamauchi, A., Tatsumi, J.    Regulation of rhizosphere acidification by photosynthetic activity    in cowpea (Vigna unguiculata L. walp.) seedlings. Ann Bot. 89,    213-220 (2002).-   18. Miller, A. J., Smith, S. J., Theodoulou, F. L. The heterologous    expression of H⁺-coupled transporters in Xenopus oocytes. In    Membrane Transport Plants and Fungi: Molecular Mechanisms and    Control (Blatt, M. R., Leigh, R. A. Sanders, D. eds) The Company of    Biologists Ltd., Cambridge, UK, pp. 167-178 (1994).-   19. Bröer, S. et al. Characterization of the monocarboxylate    transporter 1 expressed in Xenopus laevis oocytes by changes in    cytosolic pH. Biochem 1 333, 167-174 (1998).-   20. Kurschat, C. E. et al. Alkaline-shifted pH₀ sensitivity of    AE2c1-mediated anion exchange reveals novel regulatory determinants    in the AE2 N-terminal cytoplasmic domain. J Biol Chem. 281,    1885-1896 (2006).-   21. Foyer, C. H., Bloom, A. J., Queval, G., Noctor, G.    Photorespiratory Metabolism: Genes, Mutants, Energetics, and Redox    Signaling. Annu Rev Plant Biol. 60, 455-484 (2009).-   22. Kebeish, R. et al. Chloroplastic photorespiratory bypass    increases photosynthesis and biomass production in Arabidopsis    thaliana. Nat Biotechnol. 25, 593-599 (2007).-   23. Kosegarten, H., Grolig, F., Wieneke, J., Wilson, G. Hoffmann B.    Differential ammonia-elicited changes of cytosolic pH in root hair    cells of rice and maize as monitored by 2′,7′-bis-(2-carboxyethyl)-5    (and -6)-carboxyfluoresce in -fluorescence ratio. Plant Physiol.    113, 451-461 (2007).-   24. Raven, J. & Smith, F. Nitrogen assimilation and transport in    vascular land plants in relation to intracellular pH regulation. New    Phytol. 76, 415-431 (1976).-   25. Curie, C. & Briat, J. F. Iron transport and signaling in plants.    Ann Rev Plant Biol. 54, 183-206 (2003).-   26. Bellati, J. et al. Intracellular pH sensing is altered by plasma    membrane PIP aquaporin co-expression. Plant Mol Biol. 74, 105-118    (2010).-   27. Rachmilevitch, S., Cousins, A. B., Bloom, A. J. Nitrate    assimilation in plant shoots depends on photorespiration. Proc Nat    Acad Sci USA. 101, 11506-11510 (2004).-   28. Stitt, M. et al. Steps towards an integrated view of nitrogen    metabolism. J Exp Bot. 53, 959-970 (2002).-   29. Backhausen, J. E., Kitzmann, C., Scheibe, R. Competition between    electron acceptors in photosynthesis—regulation of the malate valve    during CO₂ fixation and nitrite reduction. Photosynth Res. 42, 75-86    (1994).-   30. Yamaya, T. et al. Genetic manipulation and quantitative-trait    loci mapping for nitrogen recycling in rice. J. Exp Bot. 53, 917-925    (2002).-   31. Good, A. G. et al. Engineering nitrogen use efficiency with    alanine aminotransferase. Can. J. Bot. 85, 252-262 (2007).-   32. Shrawat, A. K., Carroll, R. T., DePauw, M., Taylor, G. J.,    Good, A. G. Genetic engineering of improved nitrogen use efficiency    in rice by the tissue-specific expression of alanine    aminotransferase. Plant Biotech J. 6, 722-732 (2008).-   33. Ai, P. et al. Two rice phosphate transporters, OsPht1;2 and    OsPht1;6, have different functions and kinetic properties in uptake    and translocation. Plant J. 57, 798-809 (2009).-   34. Fan, X. R. et al. Comparing nitrate storage and remobilization    in two rice cultivars that differ in their nitrogen use efficiency.    J Exp Bot. 58, 1729-1740 (2007).-   35. Ye, J. et al. A monoclonal-antibody-based ELISA for the    detection of human FADD (Fas-associated death domain). Biotechnol    Appl Biochem. 50, 143-146 (2008).-   36. Shanks, J. H., Lappin, T. R., Hill, C. M. In situ hybridization    for erythropoietin messenger RNA using digoxigenin-labeled    oligonucleotides. Ann N Y Acad Sci. 718, 362-365 (1994).-   37. Tong, Y. P., Zhou, J. J., Li, Z., Miller, A. J. A two-component    high affinity nitrate uptake system in barley. Plant J. 41, 442-450    (2005).-   38. Orsel, M. et al. Characterization of a two component nitrate    transport and signalling high affinity nitrate uptake system in    Arabidopsis; physiology and protein—protein interaction. Plant    Physiol. 142, 1304-1317 (2006).-   39. Ho, C. H., Lin, S. H., Hu, H. C., Tsay, Y. F. CHL1 functions as    a nitrate sensor in plants. Cell 138, 1184-94 (2009).-   40. Li, C., Wong, W. H. Model based analysis of oligonucleotide    arrays: Expression index computation and outlier detection. Proc    Natl Acad Sci USA. 98, 31-36 (2001).-   41. Li, Y., Gao, Y., Xu, X., Shen, Q., Guo, S. Light-saturated    photosynthetic rate in high-nitrogen rice (Oryza sativa L.) leaves    is related to chloroplastic CO₂ concentration. J Exp Bot. 60,    2351-2360 (2009).-   42. Clough S, Bent A (1998) Floral dip: a simplified method for    Agrobacterium-mediated transformation of Arabidopsis. Plant J. 16:    735-743-   43. Orsel M, Chopin F, Leleu O, Smith S J, Krapp A, Daniel-Vedele F,    Miller A J (2006) Characterization of a Two-Component High-Affinity    Nitrate Uptake System in Arabidopsis. Physiology and Protein-Protein    Interaction. PLANT PHYSIOLOGY 142: 1304-1317-   44. Cao A, et al. Serine/threonine kinase gene Stpk-V, a key member    of powdery mildew resistance gene Pm21, confers powdery mildew    resistance in wheat. Proc Natl Acad Sci USA. 2011 10;    108(19):7727-32.-   45. Hirel et al The challenge of improving nitrogen use efficiency    in crop plants: towards a more central role for genetic variability    and quantitative genetics within integrated approaches. Journal of    Experimental Botany, Vol. 58, No. 9, pp. 2369-2387, 2007-   46. Saha et al. 1994 Planta 226: 429-442. Fan S-C, Lin C-S, Hsu P-K,    Lin S-H, Tsay Y-F (2009) The Arabidopsis Nitrate Transporter NRT1.7,    Expressed in Phloem, Is Responsible for Source-to-Sink    Remobilization of Nitrate. The Plant Cell 21: 2750-2761-   47. Jia H, Ren H, Gu M, Zhao J, Sun S, Zhang X, Chen J, Wu P, Xu    G (2011) The phosphate transporter gene OsPht1;8 is involved in    phosphate homeostasis in rice. Plant Physiol 156: 1164-1175.-   48. Kronzucker et al. Nitrate-Ammonium Synergism in Rice. A    Subcellular Flux Analysis1. Plant Physiology, March 1999, Vol. 119,    pp. 1041-1045,1999-   49. Almagro A, Lin S H, Tsay Y F (2008) Characterization of the    Arabidopsis nitrate transporter NRT1.6 reveals a role of nitrate in    early embryo development. Plant Cell 20: 3289-3299-   50. Chiu C C, Lin C S, Hsia A P, Su R C, Lin H L, Tsay Y F (2004)    Mutation of a nitrate transporter, AtNRT1:4, results in a reduced    petiole nitrate content and altered leaf development. Plant Cell    Physiol 45: 1139-1148-   51. Crawford N M, Glass A D M (1998) Molecular and physiological    aspects of nitrate uptake in plants. Trends Plant Sci 3: 389-395-   52. Fan S C, Lin C S, Hsu P K, Lin S H, Tsay Y F (2009) The    Arabidopsis nitrate transporter NRT1.7, expressed in phloem, is    responsible for source-tosink remobilization of nitrate. Plant Cell    21: 2750-2761-   53. Fan X, Gordon-Weeks R, Shen Q, Miller A J (2006) Glutamine    transport and feedback regulation of nitrate reductase activity in    barley roots leads to changes in cytosolic nitrate pools. J Exp Bot    57: 1333-1340-   54. Forde B G (2000) Nitrate transporters in plants: structure,    function and regulation. Biochim Biophys Acta 1465: 219-235-   55. Gojon A, Krouk G, Perrine-Walker F, Laugier E (2011) Nitrate    transceptor(s) in plants. J Exp Bot 62: 2299-2308-   56. Huang N C, Liu K H, Lo H J, Tsay Y F (1999) Cloning and    functional characterization of an Arabidopsis nitrate transporter    gene that encodes a constitutive component of low-affinity uptake.    Plant Cell 11: 1381-1392-   57. Kirk G J D (2003) Rice root properties for internal aeration and    efficient nutrient acquisition in submerged soil. New Phytol 159:    185-194-   58. Kronzucker H J, Glass A D M, Siddiqi M Y, Kirk G J D (2000)    Comparative kinetic analysis of ammonium and nitrate acquisition by    tropical lowland rice: implications for rice cultivation and yield    potential. New Phytol 145: 471-476-   59. Li B Z, Xin W J, Sun S B, Shen Q R, Xu G H (2006) Physiological    and molecular responses of nitrogen-starved rice plants to re-supply    of different nitrogen sources. Plant Soil 287: 145-159-   60. Li J Y, Fu Y L, Pike S M, Bao J, Tian W, Zhang Y, Chen C Z,    Zhang Y, Li H M, Huang J, et al (2010) The Arabidopsis nitrate    transporter NRT1.8 functions in nitrate removal from the xylem sap    and mediates cadmium tolerance. Plant Cell 22: 1633-1646-   61. Lin C M, Koh S, Stacey G, Yu S M, Lin T Y, Tsay Y F (2000)    Cloning and functional characterization of a constitutively    expressed nitrate transporter gene, OsNRT1, from rice. Plant Physiol    122: 379-388-   62. Lin S H, Kuo H F, Canivenc G, Lin C S, Lepetit M, Hsu P K,    Tillard P, Lin H L, Wang Y Y, Tsai C B, et al (2008) Mutation of the    Arabidopsis NRT1.5 nitrate transporter causes defective    root-to-shoot nitrate transport. Plant Cell 20: 2514-2528-   63. Liu K H, Tsay Y F (2003) Switching between the two action modes    of the dual-affinity nitrate transporter CHL1 by phosphorylation.    EMBO J 22: 1005-1013-   64. Miller A J, Fan X, Orsel M, Smith S J, Wells D M (2007) Nitrate    transport and signalling. J Exp Bot 58: 2297-2306-   65. Miller A J, Fan X, Shen Q, Smith S J (2008) Amino acids and    nitrate as signals for the regulation of nitrogen acquisition. J Exp    Bot 59: 111-119-   66. Okamoto M, Kumar A, Li W B, Wang Y, Siddiqi M Y, Crawford N M,    Glass A D M (2006) High-affinity nitrate transport in roots of    Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1.    Plant Physiol 140:1036-1046-   67. Orsel M, Krapp A, Daniel-Vedele F (2002) Analysis of the NRT2    nitrate transporter family in Arabidopsis: structure and gene    expression. Plant Physiol 129: 886-896-   68. Tsay Y F, Chiu C C, Tsai C B, Ho C H, Hsu P K (2007) Nitrate    transporters and peptide transporters. FEBS Lett 581: 2290-2300-   69. Wang Y Y, Tsay Y F (2011) Arabidopsis nitrate transporter NRT1.9    is important in phloem nitrate transport. Plant Cell 23: 1945-1957-   70. Xu G, Fan X, Miller A J (2012) Plant nitrogen assimilation and    use efficiency. Annu Rev Plant Biol 63: 153-182-   71. Yong Z, Kotur Z, Glass A D M (2010) Characterization of an    intact two component high-affinity nitrate transporter from    Arabidopsis roots. Plant J 63: 739-748-   72. Zhou J J, Fernandez E, Galván A, Miller A J (2000) A high    affinity nitrate transport system from Chlamydomonas requires two    gene products. FEBS Lett 466: 225-227-   73. Zhuo D G, Okamoto M, Vidmar J J, Glass A D M (1999) Regulation    of a putative high-affinity nitrate transporter (Nrt2; 1At) in roots    of Arabidopsis thaliana. Plant J 17: 563-568

The invention is further described by the following numbered paragraphs:

1. A method for increasing growth, yield, nitrogen use efficiency,nitrogen transport, nitrogen stress tolerance, pathogen resistance,survival and/or nitrogen acquisition of a plant comprising introducingand expressing a nucleic acid construct comprising a nucleic acidsequence as defined in SEQ ID No. 1, a functional variant or homologthereof operably linked to a regulatory sequence in a plant wherein ifthe nucleic acid sequence is as defined in SEQ ID No. 1, said plant isnot rice.

2. A method according to paragraph 1 wherein said regulatory sequence isa constitutive or strong promoter directing overexpression of saidnucleic acid.

3. A method according to paragraph 2 wherein said constitutive or strongpromoter is selected from CaMV-35S, CaMV-35Somega, Arabidopsis ubiquitinUBQ1.

4. A method according to paragraph 1 wherein said regulatory sequence isa phloem specific promoter.

5. A method according to paragraph 4 wherein said phloem specificpromoter comprises a nucleic acid comprising SEQ ID No. 5.

6. A method for making a transgenic plant having increased growth,yield, nitrogen transport, nitrogen acquisition, nitrogen stresstolerance and/or nitrogen use efficiency comprising

a) introducing and expressing in a plant or plant cell a nucleic acidconstruct comprising a nucleic acid sequence as defined in SEQ ID No. 1,a functional variant or homolog thereof operably linked to a regulatorysequence wherein if the nucleic acid sequence is as defined in SEQ IDNo. 1, said plant is not rice.

7. A method according to any of paragraphs 1 to 6 wherein said plant isa crop plant or a biofuel plant.

8. A method according to paragraph 7 wherein said crop plant is selectedfrom maize, wheat, tobacco, oilseed rape, sorghum, soybean, potato,tomato, grape, barley, pea, bean, field bean, lettuce, cotton, sugarcane, sugar beet, broccoli or other vegetable brassicas or poplar.

9. A plant obtained or obtainable from a method as defined in any ofparagraphs 6 to 8.

10. A transgenic plant expressing a nucleic acid construct comprising anucleic acid sequence as defined in SEQ ID No. 1, a functional variantor homolog thereof operably linked to a regulatory sequence if thenucleic acid sequence is as defined in SEQ ID No. 1, said plant is notrice.

11. A plant according to paragraph 9 or 10 wherein said regulatorysequence is a constitutive or strong promoter directing overexpressionof said nucleic acid.

12. A plant according to paragraph 11 wherein said constitutive promoteror strong is selected from CaMV-35S, CaMV-35Somega, Arabidopsisubiquitin UBQ1.

13. A plant according to any of paragraphs 9 or 10 wherein saidregulatory sequence is a phloem specific promoter.

14. A plant according to paragraph 13 wherein said phloem specificpromoter comprises a nucleic acid comprising SEQ ID No. 5.

15. A plant according to any of paragraphs 9 to 14 wherein said plant isa crop plant a biofuel plant.

16. A plant according to paragraph 15 wherein said crop plant isselected from maize, wheat, oilseed rape, tobacco, sorghum, soybean,potato, tomato, grape, barley, pea, bean, field bean, lettuce, cotton,sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.

17. A method for regulating pH homeostasis comprising introducing andexpressing a nucleic acid construct comprising a nucleic acid sequencecomprising SEQ ID No. 1, a functional variant or homolog thereofoperably linked to a regulatory sequence in a plant.

18. A method for reducing acidification in a plant comprisingintroducing and expressing a nucleic acid construct comprising a nucleicacid sequence comprising SEQ ID No. 1, a functional variant or homologthereof operably linked to a regulatory sequence in a plant.

19. A method for altering nitrate transport and pH homeostasis in aplant comprising introducing and expressing a nucleic acid constructcomprising a nucleic acid sequence comprising SEQ ID No. 1, a functionalvariant or homolog thereof operably linked to a regulatory sequence in aplant wherein said nucleic acid comprises a mutation in the pH sensingmotif VYEAIHKI (SEQ ID No. 16).

20. The use of a nucleic acid with homology to SEQ ID No. 1, afunctional variant or homolog thereof comprising the pH sensing motifVYEAIHKI (SEQ ID No. 16) in regulating pH in altering nitrate transportand pH homeostasis in a plant.

21. A method for increasing growth, yield, nitrogen use efficiency,nitrogen transport, pathogen resistance, survival, nitrogen stresstolerance and/or nitrogen acquisition of a plant comprising introducingand expressing a nucleic acid construct comprising a nucleic acidsequence as defined in SEQ ID No. 1, a functional variant or homologthereof operably linked to a regulatory sequence into a plant whereinsaid regulatory sequence is a constitutive promoter or a phloem specificpromoter and wherein said plant does not overexpress a nucleic acidsequence comprising SEQ ID No. 2.

22. A method for making a transgenic plant having increased growth,yield, nitrogen transport, nitrogen acquisition, nitrogen stresstolerance and/or nitrogen use efficiency comprising

a) introducing and expressing in a plant or plant cell a nucleic acidconstruct comprising a nucleic acid sequence as defined in SEQ ID No. 1,a functional variant or homolog thereof operably linked to a regulatorysequence wherein said regulatory sequence is a constitutive promoter ora phloem specific promoter and wherein said plant does not overexpress anucleic acid sequence comprising SEQ ID No. 2.

23. A method according to any of paragraphs 21 to 22 wherein saidregulatory sequence is a constitutive or strong promoter directingoverexpression of said nucleic acid.

24. A method according to paragraph 23 wherein said constitutive orstrong promoter is selected from CaMV-35S, CaMV-35Somega, Arabidopsisubiquitin UBQ1.

25. A method according to any of paragraphs 21 to 22 wherein saidregulatory sequence is a phloem specific promoter.

26. A method according to paragraph 25 wherein said phloem specificpromoter comprises a nucleic acid comprising SEQ ID No. 5.

27. A method according to any of paragraphs 21 to 26 wherein said plantis a crop plant or a biofuel plant.

28. A method according to paragraph 27 wherein said crop plant isselected from maize, rice, wheat, oilseed rape, tobacco, sorghum,soybean, potato, tomato, grape, barley, pea, bean, field bean, lettuce,cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas orpoplar.

29. A method according to paragraph 28 wherein said crop plant is notrice.

30. A plant obtained or obtainable from a method as defined in any ofparagraphs 21 to 29.

31. A transgenic plant expressing a nucleic acid construct comprising anucleic acid sequence as defined in SEQ ID No. 1, a functional variantor homolog thereof operably linked to a regulatory sequence into a plantwherein said regulatory sequence is a constitutive promoter or a phloemspecific promoter and wherein said plant does not overexpress a nucleicacid sequence comprising SEQ ID No. 2.

32. A plant according to paragraph 30 or 31 wherein said regulatorysequence is a constitutive or strong promoter directing overexpressionof said nucleic acid.

33. A plant according to paragraph 32 wherein said constitutive promoteror strong is selected from CaMV-35S, CaMV-35Somega, Arabidopsisubiquitin UBQ1.

34. A plant according to any of paragraphs 30 to 31 wherein saidregulatory sequence is a phloem specific promoter.

35. A plant according to paragraph 34 wherein said phloem specificpromoter comprises a nucleic acid comprising SEQ ID No. 5.

36. A plant according to any of paragraphs 31 to 35 wherein said plantis a crop plant or biofuel plant.

37. A plant according to paragraph 36 wherein said crop plant isselected from maize, rice, wheat, oilseed rape, sorghum, soybean,potato, tomato, grape, barley, pea, bean, field bean, lettuce, cotton,sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.

38. A plant according to paragraph 37 wherein said crop plant is notrice.

39. A product derived from a plant as defined in any of paragraphs 9 to16 or 31 to 38.

* * *

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A method for increasing growth, yield, nitrogenuse efficiency, nitrogen transport, nitrogen stress tolerance, pathogenresistance, survival and/or nitrogen acquisition of a plant comprisingintroducing and expressing a nucleic acid construct comprising a nucleicacid sequence encoding a nitrate transporter having pH sensing motifcomprising VYEAIHKI and encoding a polypeptide comprising SEQ ID NO: 3or homolog thereof, operably linked to a regulatory sequence in a plantwherein if the polypeptide encoded comprises SEQ ID NO: 3, said plant isnot rice.
 2. A method according to claim 1, wherein said regulatorysequence is a constitutive promoter directing overexpression of saidnucleic acid.
 3. A method according to claim 1, wherein said regulatorysequence is a phloem specific promoter.
 4. A method for making atransgenic plant having increased growth, yield, nitrogen transport,nitrogen acquisition, nitrogen stress tolerance and/or nitrogen useefficiency comprising a) introducing and expressing in a plant or plantcell a nucleic acid construct comprising a nucleic acid sequenceencoding a nitrate transporter having pH sensing motif comprisingVYEAIHKI and encoding a polypeptide comprising SEQ ID NO: 3 or homologthereof, operably linked to a regulatory sequence wherein if thepolypeptide comprises SEQ ID NO: 3, said plant is not rice.
 5. A methodaccording to claim 1, wherein said plant is a crop plant or a biofuelplant or bioenergy crop, wherein the biofuel plant or bioenergy crop isselected from the group consisting of rape/canola, sugar cane, sweetsorghum, Panicum virgatum (switchgrass), linseed, lupin, willow, poplar,poplar hybrid Miscanthus or gymnosperm, loblolly pine, maize, grass. 6.The method according to claim 5, wherein said crop plant is selectedfrom maize, wheat, tobacco, oilseed rape, sorghum, soybean, potato,tomato, grape, barley, pea, bean, field bean, lettuce, cotton, sugarcane, sugar beet, broccoli or other vegetable brassicas or poplar. 7.The method according to claim 5, wherein said plant is maize or soybean.8. A method according to claim 1, wherein said plant is maize or soybeanand said homolog is from maize or soybean.
 9. A plant obtained orobtainable from a method as defined in claim 8, wherein the plantcomprises said nucleic acid construct.
 10. A transgenic plant expressinga nucleic acid construct comprising a nucleic acid sequence encoding anitrate transporter having pH sensing motif comprising VYEAIHKI andencoding a polypeptide comprising SEQ ID NO: 3 or homolog thereof,operably linked to a regulatory sequence wherein if the polypeptidecomprises SEQ ID NO: 3, said plant is not rice.
 11. The plant accordingto claim 10, wherein said regulatory sequence is a constitutive promoterdirecting overexpression of said nucleic acid.
 12. The plant accordingto claim 10, wherein said regulatory sequence is a phloem specificpromoter.
 13. The plant according to claim 10, wherein said plant is acrop plant or a biofuel plant or bioenergy crop, wherein the biofuelplant or bioenergy crop is selected from the group consisting ofrape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass),linseed, lupin, willow, poplar, poplar hybrid Miscanthus or gymnosperm,loblolly, pine, maize, grass.
 14. The plant according to claim 13,wherein said crop plant is selected from maize, wheat, oilseed rape,tobacco, sorghum, soybean, potato, tomato, grape, barley, pea, bean,field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or othervegetable brassicas or poplar.
 15. The plant according to claim 10,wherein said plant is maize or soybean.
 16. The plant according to claim10, wherein said plant is maize or soybean and said homolog is frommaize or soybean.
 17. A method for regulating pH homeostasis orreducing/altering acidification in a plant comprising introducing andexpressing a nucleic acid construct comprising a nucleic acid sequenceencoding a nitrate transporter having pH sensing motif comprisingVYEAIHKI and encoding a polypeptide comprising SEQ ID NO: 3 or homologthereof, operably linked to a regulatory sequence in a plant.
 18. Themethod according to claim 17, wherein said plant is selected from maize,wheat, tobacco, oilseed rape, sorghum, soybean, potato, tomato, grape,barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet,broccoli or other vegetable brassicas or poplar.
 19. The methodaccording to claim 17, wherein said plant is maize or soybean.
 20. Themethod according to claim 17, wherein said plant is maize or soybean andwherein said homolog is from maize or soybean.
 21. The method accordingto claim 17, wherein said regulatory sequence is a constitutivedirecting overexpression of said nucleic acid.
 22. The method accordingto claim 17, wherein said regulatory sequence is a phloem specificpromoter.
 23. The method of claim 1, wherein said homolog comprises asequence having at least 95% identity to SEQ ID NO:
 3. 24. The method ofclaim 4, wherein said homolog comprises a sequence having at least 95%identity to SEQ ID NO:
 3. 25. The method of claim 17, said homologcomprises a sequence having at least 95% identity to SEQ ID NO:
 3. 26.The plant of claim 10, said homolog comprises a sequence having at least95% identity to SEQ ID NO:
 3. 27. The method of claim 1, wherein saidplant comprising said nucleic acid sequence has increased nitrogenupdate in the absence of a nucleic acid sequence introduced into saidplant comprising OsNAR21.
 28. The method of claim 1, wherein said plantcomprising said nucleic acid sequence has reduced acidification comparedto a plant not comprising said construct.
 29. A method for increasinggrowth, yield, nitrogen use efficiency, nitrogen transport, nitrogenstress tolerance, pathogen resistance, survival and/or nitrogenacquisition of a plant comprising introducing and expressing a nucleicacid construct comprising a nucleic acid encoding a nitrate transporterhaving pH sensing motif comprising VYEAIHKI and encoding a polypeptidecomprising SEQ ID NO: 3 or a sequence having at least 95% identity toSEQ ID NO: 3, operably linked to a regulatory sequence in a plantwherein if the polypeptide encoded comprises SEQ ID NO: 3, said plant isnot rice.